Isolation of proteins

ABSTRACT

The present invention relates to a novel method for the isolation or purification of immunoglobulins (a special class of proteins) from a solution containing immunoglobulins, e.g. hybridoma cell culture supernatants, animal plasma or sera, or colostrum. The method includes the use of a minimum of salts, such as lyotropic salts, in the binding process and preferably also the use of small amounts of organic solvents in the elution process. The solid phase matrices, preferably epichlorohydrin activated agarose matricees, are functionalised with mono- or bicyclic aromatic or heteroaromatic ligands (molecular weight: at the most 500 Dalton) which, preferably, comprises an acidic substituent, e.g. a carboxylic acid. The matrices utilised show excellent properties in a “Standard Immunoglobulin Binding Test” and in a “Monoclonal Antibody Array Binding Test” with respect to binding efficiency and purity, and are stable in 1M NaOH.

This application is a Continuation of Application Ser. No. 10/268,667filed Oct. 11, 2002, which issued as U.S. Pat. No. 6,919,436, and whichis a Divisional of Application Ser. No. 09/147,668 filed Mar. 19, 1999,which issued as U.S. Pat. No. 6,498,236 and which is a National PhaseApplication of PCT application Ser. No. PCT/DK97/00359 filed Sep. 1,1997, which claims priority to Denmark Application Serial No. 0932/96filed Aug. 30, 1996.

FIELD OF THE INVENTION

The present invention relates to a method for isolation or purificationof immunoglobulins from various raw materials and solid phase matricestherefor.

BACKGROUND OF THE INVENTION

Immunoglobulins—or antibodies—constitute a very important class ofproteins which are present in various body fluids of mammals, birds andfish functioning as protective agents of the animal against substances,bacteria and virus challenging the animal. Immunoglobulins are typicallypresent in animal blood, milk, and saliva as well as other body fluidsand secretions.

The biological activity, which the immunoglobulins possess, is todayexploited in a range of different applications in the human andveterinary diagnostic, health care and therapeutic sector.

Diagnostics

Antibodies have for many years been applied as an important analytictool in connection with detection and quantification of a large varietyof substances of relevance in the diagnosis of diseases and areincreasingly important in areas such as quality control of foodproducts, environmental control, drugs of abuse, and monitoring andcontrol of industrial processes.

For these purposes, the desired antibodies can be produced byhyper-immunisation of suitable host animals, such as rabbits and sheep,or, alternatively, by producing monoclonal antibodies in hybridoma cellcultures.

Following the primary production of the antibodies in either a hostanimal or in cell culture, the antibody is typically isolated from thebulk of other substances in the raw material by some sort of isolationprocess. This is necessary in order to avoid interference from theseother substances with the antibody activity in the analyticalapplication.

Health Care and Therapeutic Applications

Passive immunisation by intramuscularly injection of immunoglobulinconcentrates is a well-known application for temporary protectionagainst infectious diseases, which is typically applied when people aretravelling from one part of the world to the other. The success of thiskind of treatment on humans is now being followed up in the veterinaryfield where passive immunisation of new born cattle, horses, pigs andchickens are being applied and developed to enhance the survival rate ofthese animals during their first weeks of live. An important issue inthis field is of course the cost of such a treatment, which to a highdegree depends on the cost of producing the immunoglobulin concentrate.

Isolates of animal immunoglobulins, e.g. from bovine milk, are alsounder investigation as an oral health care or even therapeutic productto avoid or treat gastrointestinal infections, e.g. in AIDS patients.For such applications both the degree of purity of the product as wellas the cost is of major importance.

A more sophisticated application of antibodies for therapeutic use isbased on so called “drug-targeting” where very potent drugs arecovalently linked to antibodies with specific binding affinities towardsspecific cells in the human organism, e.g. cancer cells. This techniqueensures that the drug is concentrated on the diseased cells givingmaximal effect of the drug without the severe side-effects thatfrequently occurs when using chemotherapy. For such purposes theantibodies have to be very carefully controlled and of high purity, andthe typical way of performing the primary production are either byproducing monoclonal antibodies in hybridoma cell culture or byfermenting genetically engineered bacteria, e.g. E. coli.

Isolation of Immunoglobulins

All the above mentioned applications of immunoglobulins requires somesort of isolation of the antibody from the crude raw material, but eachkind of application has its own very varying demands with respect to thefinal purity and allowable cost of the antibody product.

Generally, there exists a very broad range of different methodsavailable for isolation of immunoglobulins giving a very broad range offinal purities, yields and cost of the product.

Traditional methods for isolation of immunoglobulins are based onselective reversible precipitation of the protein fraction comprisingthe immunoglobulins while leaving other groups of proteins in solution.Typical precipitation agents being ethanol, polyethylene glycol,lyotropic (anti-chaotropic) salts such as ammonium sulfate and potassiumphosphate, and caprylic acid.

Typically, these precipitation methods are giving very impure productswhile at the same time being time consuming and laborious. Furthermore,the addition of the precipitating agent to the raw material makes itdifficult to use the supernatant for other purposes and creates adisposal problem. This is particularly relevant when speaking of largescale purification of immunoglobulins from, e.g., whey and plasma.

Ion exchange chromatography is another well known method of proteinfractionation frequently used for isolation of immunoglobulins. However,this method is not generally applicable because of the restraints inionic strength and pH necessary to ensure efficient binding of theantibody together with the varying isoelectric points of differentimmunoglobulins.

Protein A and Protein G affinity chromatography are very popular andwidespread methods for isolation and purification of immunoglobulins,particularly for isolation of monoclonal antibodies, mainly due to theease of use and the high purity obtained. Although being popular it ishowever recognised that Protein A and Protein G poses several problemsto the user among which are: very high cost, variable binding efficiencyof different monoclonal antibodies (particularly mouse IgG₁), leakage ofProtein A/Protein G into the product, and low stability of the matrix intypical cleaning solutions, e.g. 1 M sodium hydroxide. Each of thesedrawbacks have its specific consequence in the individual application,ranging from insignificant to very serious and prohibitive consequences.

Hydrophobic chromatography is also a method widely described forisolation of immunoglobulins, e.g. in “Application Note 210, BioProcessMedia” published by Pharmacia LKB Biotechnology, 1991. In this referencea state of the art product “Phenyl Sepharose High Performance” isdescribed for the purpose of purifying monoclonal antibodies from cellculture supernatants. As with other hydrophobic matrices employed so farit is necessary to add lyotropic salts to the raw material to make theimmunoglobulin bind efficiently. The bound antibody is released from thematrix by lowering the concentration of lyotropic salt in a continuousor stepwise gradient. It is recommended to combine the hydrophobicchromatography with a further step if highly pure product is the object.

The disadvantage of this procedure is the necessity to add lyotropicsalt to the raw material as this gives a disposal problem and therebyincreased cost to the large scale user. For other raw materials thancell culture supernatants such as whey, plasma, and egg yolk theaddition of lyotropic salts to the raw materials would in many instancesbe prohibitive in large scale applications as the salt would prevent anyeconomically feasible use of the immunoglobulin depleted raw material incombination with the problem of disposing several thousand litres ofwaste.

Thiophilic adsorption chromatography was introduced by J. Porath in 1985(J. Porath et al; FEBS Letters, vol. 185, p. 306, 1985) as a newchromatographic adsorption principle for isolation of immunoglobulins.In this paper, it is described how divinyl sulfone activated agarosecoupled with various ligands comprising a free mercapto-group showspecific binding of immunoglobulins in the presence of 0.5 M potassiumsulfate, i.e. a lyotropic salt. It was postulated that the sulfonegroup, from the vinyl sulfone spacer, and the resulting thio-ether inthe ligand was a structural necessity to obtain the describedspecificity and capacity for binding of antibodies. It was however latershown that the thio-ether could be replaced by nitrogen or oxygen if theligand further comprised an aromatic radical (K. L. Knudsen et al,Analytical Biochemistry, vol 201, p.170, 1992).

Although the matrices described for thiophilic chromatography generallyshow good performance, they also have a major disadvantage in that it isneeded to add lyotropic salts to the raw material to ensure efficientbinding of the immunoglobulin, which is a problem for the reasonsdiscussed above.

Other thiophilic ligands coupled to epoxy activated agarose have beendisclosed in (J. Porath et. al., Makromol. Chem., Makromol. Symp., vol.17, p.359, 1988) and (A. Schwarz et. al., Journal of Chromatography B,vol.664, pp.83-88, 1995), e.g. 2-mercaptopyridine, 2-mercaptopyrimidine,and 2-mercaptothiazoline. However, all these affinity matrices stillhave inadequate affinity constants to ensure an efficient binding of theantibody without added lyotropic salts.

Binding and Isolation of Proteins and Other Biomolecules

WO 96/00735 and WO 96/09116 disclose resins (matrices) for purifyingproteins and peptides which resins are characterised by the fact thatthey contain ionizable ligands and/or functionalities which areuncharged at the pH of binding the target protein or peptide, therebyfacilitating hydrophobic interactions, and charged at the pH ofdesorption, thereby disrupting the established hydrohobic interactionbetween the resin and the target protein or peptide. WO 96/00735mentions the possibility of coupling 2-mercapto-benzimidazole toepoxy-activated Sepharose 6 B. The actual ligand concentration is notdisclosed, however the coupling was performed with an epoxy-activatedSepharose wherein the content of epoxy-groups is disclosed to be in therange of 1.02-1.28 mmol/g dry matter.

WO 92/16292 discloses a number of different ligands coupled to divinylsulfone activated agarose and the use of the resulting solid phasematrices for thiophilic adsorption of proteins, preferablyimmunoglobulins. Specifically is mentioned solid phase matricescomprising 4-amino-benzoic acid as a ligand on a divinyl sulfoneactivated agarose. The adsorption of proteins, preferablyimmunoglobulins in WO 92/16292, is performed at high concentrations oflyotropic salts i.e. with an ionic strength of on or above 2.25.

BRIEF DESCRIPTION OF THE INVENTION

It has now surprisingly been found that several types of aromatic orheteroaromatic substances linked to a solid phase matrix can be utilisedin a novel method for the isolation and/or purification ofimmunoglobulins of different kinds from widely different raw materialswith high efficiency and with special advantages with respect to the useof little or no salts, especially lyotropic salts, in the bindingprocess and with respect to the ability to bind a wide range ofimmunoglobulins. Furthermore, these matrices have special advantageswith respect to stability in NaOH, which is especially relevant when thesolid phase matrices are to be regenerated after use.

Thus, an object of the present invention is to provide a method for theisolation of immunoglobulins from a solution containing one or moreimmunoglobulins, comprising the following operations:

-   a) contacting a solution containing one or more immunoglobulins and    having a pH in the range of 2.0 to 10.0 and a total salt content    corresponding to a ionic strength of at the most 2.0 with a solid    phase matrix of the general formula    M-SP1-L,    -   wherein M designates the matrix backbone, SP1 designates a        spacer, and L designates a ligand comprising a mono- or bicyclic        optionally substituted aromatic or heteroaromatic moiety,        whereby at least a part of the immunoglobulins becomes bound to        the solid phase matrix;-   b) separating the solid phase matrix having immunoglobulins bound    thereto from the solution;-   c) optionally washing the solid phase matrix, and-   d) contacting the solid phase matrix with an eluent in order to    liberate the one or more immunoglobulins from the solid phase    matrix;    with the first proviso that at least two of the criteria (a), (b),    and (c) are fulfilled:    -   (a) the solid phase matrix has a binding efficiency of at least        50% when tested at a pH in the range of 2.0 to 10.0 in the        “Standard Immunoglobulin Binding Test” described herein; or    -   (b) the solid phase matrix has an average binding efficiency of        at least 60% for all of the immunoglobulins tested in the        “Monoclonal Antibody Array Binding Test” when tested at a pH in        the range of 2.0 to 10.0; or    -   (c) the stability of the solid phase matrix in 1 M NaOH is so        that incubation of the matrix in 1 M NaOH in the dark at room        temperature for 7 days reduces the binding efficiency at a pH in        the range of one pH unit lower than the binding maximum pH value        to one pH unit higher than the binding maximum pH value, as        determined in the “Standard Immunoglobulin Binding Test”        described herein, with less than 25% compared to a corresponding        untreated matrix; and        with the second proviso that the molecular weight of the ligand        -L is at the most 500 Dalton.

The present invention furthermore provides a solid phase matrix,comprising a functionalised matrix backbone carrying a plurality offunctional groups of the following formulaM-SP1-L

-   -   wherein M designates the matrix backbone, SP1 designates a        spacer, and L designates a ligand comprising a mono- or bicyclic        optionally substituted aromatic or heteroaromatic moiety,        and wherein at least two of the criteria (a), (b), and (c) are        fulfilled:    -   (a) the solid phase matrix has a binding efficiency of at least        50% when tested at a pH in the range of 2.0 to 10.0 in the        “Standard Immunoglobulin Binding Test” described herein; or    -   (b) the solid phase matrix has a binding efficiency of at least        40% for all of the immunoglobulins tested in the “Monoclonal        Antibody Array Binding Test” when tested at a pH in the range of        2.0 to 10.0; or    -   (c) the stability of the solid phase matrix in 1 M NaOH is so        that incubation of the matrix in 1 M NaOH in the dark at room        temperature for 7 days reduces the binding efficiency at a pH in        the range of one pH unit lower than the binding maximum pH value        to one pH unit higher than the binding maximum pH value, as        determined in the “Standard Immunoglobulin Binding Test”        described herein, with less than 25% compared to a corresponding        untreated matrix;        with the first proviso that the molecular weight of the ligand        -L is at the most 500 Dalton; and with the second proviso that        when M is agarose and SP1 is derived from vinyl sulfone then L        is not 4-aminobenzoic acid,        which is especially suited for use in the method according to        the invention.

It has furthermore been found that the matrices mentioned above, whereinthe aromatic or heteroaromatic moiety is carrying an acidic group,optionally via a spacer SP2, are equally suited for the isolation andpurification of proteins without the need to add lyotropic salts to theprotein containing solution (the raw material) and without the need touse large amounts of organic solvents for elution of the bound proteinsfrom the matrix.

Thus, the present invention also provides a solid phase matrix,comprising a functionalised matrix backbone carrying a plurality offunctional groups of the following formulaM-SP1-X-A-SP2-ACID

-   -   wherein M designates the matrix backbone; SP1 designates a        spacer; X designates —O—, —S—, or NH—; A designates a mono- or        bicyclic optionally substituted aromatic or heteroaromatic        moiety; SP2 designates an optional spacer; and ACED designates        an acidic group;        with the first proviso that the molecular weight of the ligand        -L is at the most 500 Dalton; and with the second proviso that        when M is agarose and SP1 is derived from vinyl sulfone then L        is not 4-aminobenzoic acid;        and a method for the isolation of proteins from a solution        containing one or more of proteins, comprising the following        operations:

-   a) contacting a solution containing one or more proteins having a pH    in the range of 1.0 to 6.0 and a total salt content corresponding to    an ionic strength of at the most 2.0 with a solid phase matrix as    described herein, where by at least a part of the proteins becomes    bound to the solid phase matrix;

-   b) separating the solid phase matrix having proteins bound thereto    from the solution;

-   c) optionally washing the solid phase matrix; and

-   d) contacting the solid phase matrix with an eluent in order to    liberate one or more of the proteins from the solid phase matrix,    wherein the eluent used comprises less than 10% (v/v) of organic    solvents.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of Immunoglobulins

In general, the method for isolation of immunoglobulins may be dividedinto several steps:

-   (a) Equilibration of the solid phase matrix-   (b) Contacting the slid phase with immunoglobulin solution-   (c) Washing the solid phase-   (d) Separation of the solid phase from the solution-   (d) Elation of the bound immunoglobulin-   (e) Regeneration of the solid phase matrix

It may however depend on the specific application whether all steps areperformed each time or at all. Thus, the only mandatory steps are thecontacting, separation, and the elution steps, while the equilibration,washing, and regeneration steps may or may not be performed according tothe specific requirements relevant to the actual application. The typeof the separation step depends on the actual set-up (see below).

Equilibration

Before contacting the solid phase matrix with the immunoglobulincontaining solution it is preferred to ensured that both the matrix andthe solution are in a condition resulting in the wanted binding ofimmunoglobulin. In this respect, it may therefore be necessary to adjustparameters such as pH, ionic strength, and temperature and in someinstances the addition of substances of different kind to promotebinding of immunoglobulins and/or to prevent binding of impurities.

Thus, it is an optional step to perform an equilibration of the solidphase matrix by washing it with a solution (e.g. a buffer for adjustingpH, ionic strength, etc., or for the introduction of a detergent)bringing the necessary characteristics to the solid phase.

Contacting

When the solid phase matrix is in the form of particles of eitherspherical or irregular form the contacting of a solution containing oneor more immunoglobulins may be performed either in a packed bed columnor in a fluidised/expanded bed column containing the solid phase matrix.It may also be performed in a simple batch operation where the solidphase matrix is mixed with the solution for a certain time to allowbinding of the immunoglobulin(s).

Whenever the solid phase matrix is in the form of permeable orsemi-permeable membranes or sheets the contacting is generally performedby pumping/forcing the immunoglobulin containing solution across thesurface and/or through a porous structure of the membrane or sheet toensure that the immunoglobulins are coming in close contact with theligands immobilised on the surface and/or in the porous structures.

Further guidelines for this process are given in “Purification Tools forMonoclonal Antibodies”, Gagnon, P., Validated Biosystems, 1996.

Washing

After contacting the solid phase matrix with the immunoglobulincontaining solution there is optionally performed a washing procedure toremove unbound or loosely bound substances such as other proteins,lipids, nucleic acids or other impurities from the matrix. However insome cases where very high purity of the immunoglobulin is not criticalthe washing procedure may be omitted saving a process-stop as well aswashing solution.

In other cases where very high purity of the immunoglobulin is neededthere may be employed several different washing procedures withdifferent washing buffers-before elution is commenced.

The washing buffers employed will depend on the nature of thechromatographic adsorbent and the ligand binding the immunoglobulins.The washing buffer should not disturb the binding of the immunoglobulinto the adsorbent i.e. pH, salt concentration and other additives shouldbe adjusted so that only the unwanted impurities are removed either bysimple substitution of the solution containing impurities and present inand around the adsorbent with the washing buffer—or in combinationherewith also releasing impurities bound to the adsorbent. The releasingof impurities bound to the matrix may be accomplished either by changingpH and/or ionic strength or by adding a substance to the washing bufferwhich interacts competitively with either the adsorbent or the impurity,and thereby displacing the impurity from the adsorbent.

The washing (operation (c) in the method according to the invention) ispreferably performed in order to remove remainders from the solutioncontaining the immunoglobulins, and in order to remove other type ofbiomolecules.

Elution

Elution of the bound immunoglobulin is generally performed by contactingthe solid phase matrix comprising the bound immunoglobulin with asolution that releases the immunoglobulin from the ligand on the matrix.The immunoglobulin is hereby released into the solution and can bewashed out of the matrix. The solution employed to release theimmunoglobulin should generally have different characteristics than whatwas used for binding of the immunoglobulin e.g. the solution may have adifferent pH, a different ionic strength, a different temperature and/orit may comprise organic solvents (preferably only small amounts),detergents, chaotropes or other denaturing reagents. Combinations ofchanges in these different parameters are also generally employed.

Elution may also be performed by applying a solution gradually changingthe conditions from binding to non-binding conditions, a procedure whichtypically is phrased gradient elution.

Once the immunoglobulin have been released from the solid phase matrixinto the eluting solution it may be recovered from this by differentoptional means known per se. In the most simple case the immunoglobulinmay be used directly without any changes but in many instances some sortof concentrating procedure would be preferred e.g. ultra-filtration,freeze-drying or precipitation (e.g. salting out). The immunoglobulinsolution may also very well be purified further in a further processingstep of optional character.

Regeneration

The solid phase matrix may optionally by cleaned i.e. regenerated afterelution of the immunoglobulin. This procedure is typically performedregularly to minimise the building up of impurities fouling up thesurface of the solid phase and/or to sterilise the matrix to avoidcontamination of the product with microorganisms proliferating andescaping from the solid phase and the equipment used during the process.Popular ways of performing such a regeneration step is to wash the solidphase matrix with solutions able to clean the matrix and/or killmicroorganisms. Typical solutions for these purposes would be, e.g.,0.1-1.0 M sodium hydroxide; solutions of peracids or hydrogen peroxide;denaturants such as guanidinium hydrochloride; solutions comprisingactive chlorine such as hypochlorite solutions, organic solvents such asethanol; detergents etc. An especially preferred method for this purposeis to use 0.1-1.0 M sodium hydroxide due to the very high efficiency,low cost, ease of neutralization with hydrochloric acid and lack ofwaste problems.

In a preferred embodiment of the present invention the method includes:(i) equilibration (optional step), (ii) contacting, (iii) washing(optional step), (iv) separation, (v) elution, and (vi) regeneration,where cycle of steps (i)-(v) are repeated one or several times beforeregeneration, and were the solid phase matrix is reused afterregeneration.

The conditions employed in both the binding, washing and elution step(s)may be very decisive for the resulting binding efficiency, yield andpurity of the immunoglobulin. Different solid phase matrices accordingto the invention may need different binding, washing and elutionconditions to ensure an optimal result. Likewise the nature of the rawmaterial will have a very significant impact on the conditions chosenfor that particular isolation procedure e.g. very dilute solutions ofmonoclonal antibodies in hybridoma cell culture supernatants (typically10-100 μg/ml) behave differently than the same type of antibodiespresent in more concentrated solutions such as ascites fluids (1-5mg/ml) and immunoglobulins present in, e.g., whey (1-2 mg/ml) need otherconditions than immunoglobulins from plasma/serum (5-20 mg/ml) etc.

Also the composition i.e. the contents of different types of impuritiesmay vary significantly between different raw materials, e.g., egg yolkhas a very different composition as compared to hybridoma cell culturesupernatants.

As mentioned above it is generally possible to add different substancesto the immunoglobulin containing solution as to enhance the binding ofantibodies to the solid phase matrix.

In a particular embodiment, the present invention relates to methods forthe isolation of immunoglobulins and solid phase matrices thereforyielding anisolated immunoglobulin of a purity of at least 10% such asat least 30%, preferably at least 50% such as at least 70%, morepreferably at least 80% such as 90%, in particular at least 99%.

As mentioned above, it is believed that the binding efficiency maximumpH value for the solid phase matrices is in the range of 2.0 to 10.0,most likely in the range of 3.0 to 9.0. It is therefore most relevant toconduct the isolation process near that maximum (which of course mayvary for different combinations of immunoglobulins/solid phase matices.Thus, the pH of the solution containing the immunoglobulins (or proteinsin general) is preferably in the range of 2.0 to 10, such as in therange of 3.0 to 9.0. However, depending on the ligand type and thematrix backbone, the pH range is preferably 3.0 to 7.0 or 6.0 to 9.0.

It is believed that, when the ligand is of the type -X-A-SP2-ACID, thenshould the pH of the solution containing the immunoglobulins be in therange of 2.0 to 6.0, preferably in the range of 2.5 to 5.5 such as inthe range of 3.0 to 5.5, or in the range of 4.0 to 5.5, corresponding toan expected binding efficiency maximum for that specific type of matrix.

With respect to contacting operation (a) above, it has been found thatit is not necessary to add excessive amounts of lyotropic salt in orderfor the immunoglobulins to bind to the matrix. Thus, the total saltcontent, including e.g. NaCl, of the solution containing theimmunoglobulins need only be so that it corresponds to a ionic strengthof at the most 2.0, preferably in the range of 0.05 to 2.0, such as 0.05to 1.4, especially in the range of 0.05 to 1.0. As an alternativerequirement, the concentration of lyotropic salt as such should be aslow as possible, thus, it has been shown that it is possible to operatesolution containing immunoglobulins where the concentration of lyotropicsalts is at the most 0.4 M, preferably at the most 0.3 M, in particularat the most 0.2 M, such as at the most 0.1 M.

Examples of lyotropic salts are given in “Purification Tools forMonoclonal Antibodies”, Gagnon, P., Validated Biosystems, 1996), wherethe Hofmeister series of lyotropic ions are presented.

With respect to the concentration of immunoglobulins in the solution, itis believed that the solid phase matrices can operate for a very largerange concentration range, thus, it is believed that the solid phasematrices operate equally efficient for concentration of immunoglobulinsin the solution containing the immunoglobulins in the range of 0.001 to0.2, preferably 0.01 to 0.1, mg/ml, as in hybridoma cell culturesupernatants, in the range of 0.2 to 2.0 mg/ml as in milk and whey, inthe range of 5.0 to 20 mg/ml as for different animal sear and plasma,and even in the range of 20-80 mg/ml as for colostrum.

It has been found that the present invention is especially suitable forsolutions comprising in the range of 0.1 to 30 mg immunoglobulins pergram of solid phase matrix, such as in the range of 0.2 to 2 or in therange of 5.0 to 25 mg per gram of solid phase matrix.

Thus, the solution containing the immunoglobulins may be artificially aswell as biologically solution of immunoglobulins such as crudefermentation broths; mammalian cell cultures such as hybridoma cellcultures; fermentation broths from cultures of genetically engineeredmicroorganisms such as E. coli; ascites fluids such as mouse and ratascites fluid; milk, whey, blood, plasma and serum from man, mouse, rat,cow, pig, rabbit, goat, guinea pig, and donkey; and egg yolk such aschicken egg yolk.

Furthermore, it has been shown (see the examples) that specialadvantages with respect to purity may be obtained when the solutioncontaining the immunoglobulins comprises a negatively charged detergent.Without being bound to any theory it is believed that the detergentsuppresses the adherence of other biomolecules to the matrix. Examplesof such detergent are octyl sulfate, bromphenol blue, octane sulfonate,sodium laurylsarcosinate, and hexane sulfonate.

Also, in the washing step (operation (c) of the method according to theinvention) it is, probably for the same reasons, advantageous to use annegatively charged detergent. The detergent may be used alone or incombination with an buffer, e.g. a lyotropic salt buffer. Use oflyotropic salts in the washing step (small volume) represents only aminor waste product problem compared with using lyotropic salts in thebinding processes (operation (a)) (in that the binding process includesthe use of large volumes is most cases).

Also, the excellent properties of the solid phase matrices for use inthe method according to the invention may be expressed even without theuse of organic solvents in the eluation step (operation (d)), thus,preferably, the eluent used comprises less than 10% (v/v), morepreferably less than 5%, of organic solvents. Most preferably, noorganic solvents are used at all.

Alternatively, as has been shown in example 14, a larger amount ofnon-toxic solvents, e.g. propylene glycol, may be used, e.g. up to 40%propylene glycol.

The contacting step (operation (a)) as well as the following step, i.e.separation, washing, and eluation, may be performed in various way. Thephysical measures selected are often guided by the scale and whether theprocess has to be repeated. The solid phase matrices according to theinvention may be used in almost any of the set-ups used for developmentand for industrial purposes. Thus, the solid phase matrix may becontacted with the solution containing the immunoglobulins, e.g., in astirred batch process, in a packed bed chromatographic column process,and in a fluidised bed process. Further guidelines are given in“Purification Tools for Monoclonal Antibodies”, Gagnon, P., ValidatedBiosystems, 1996.

Other necessary measures for performing the isolation of immunoglobulinsaccording to the invention follow conventional methodologies.

The present invention provides a method for the isolation andpurification of immunoglobulins from a large variety of raw materialshaving different concentrations of immunoglobulins, typically rangingfrom about 10 μg/ml in hybridoma cell culture supernatants and about 1-2mg/ml in milk and whey to about 5-20 mg/ml in different animalsera/plasma, and up to 50-60 mg/ml in colostrum. The nature and relativeconcentration of different impurities potentially interfering with thebinding and isolation of immunoglobulins are also varying to a greatextent between the different immunoglobulin sources.

For some applications of immunoglobulins it is of high important thatthe immunoglobulins are extremely pure, e.g. having a purity of morethan 99%. This is particularly true whenever the immunoglobulin is to beused as a therapeutic, but is also necessary for other applications. Inthe diagnostic field the degree of purity needed may depend on a numberof factors such as whether the antibody is to be used un-derivatised, inwhich case there may not be required a high degree of purity, i.e. lessthan 50%, or whether the antibody has to be labelled with a signalmolecule such as an enzyme, e.g. horseradish peroxidase, in which casethe antibody often is required to be at least 80% pure or more. Forother applications the need for purity may differ correspondingly. Itseems however to be a general demand that the purity of theimmunoglobulin is at least 10% on a dry matter basis to enable a properuse of the product.

However, the present invention provides, as it should be clear,guidelines for solving these problems.

Solid Phase Matrices

As described above, the method according to the invention includes theuse of a solid phase matrix, where the solid phase matrix comprises afunctionahised matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1-Lwherein M designates the matrix backbone, SP1 designates a spacer, and Ldesignates a ligand comprising an mono- or bicyclic optionallysubstituted aromatic or heteroaromatic moiety, which has to fulfilcertain criteria.

It should be noted that the present invention also relates to thesesolid phase matrices as such. Thus, the definitions below relate to themethod according to the invention as well as to the solid phase matricesaccording to the invention.

The solid phase matrix may comprise, as the matrix backbone, any naturalor synthetic and organic or inorganic material known per se to beapplicable in solid phase separation of proteins and other biomolecules,e.g. natural or synthetic polysaccharides such as agar-agar andagaroses; celluloses, cellulose ethers such as hydroxypropyl cellulose,carboxymethyl celluose; starches; gums such as guar gum, and gum arabic,gum ghatti, gum tragacanth, locust bean gum, xanthan gum; pectins;mucins; dextrans; chitins; chitosans; alginates; carrageenans; heparins;gelatins; synthetic polymers such as polyamides such as polyacrylamidesand polymethacrylamides; polyimides; polyesters; polyethers; polymericvinyl compounds such as polyvinylalcohols and polystyrenes; polyalkenes;inorganic materials such as silicious materials such as silicon dioxideincluding amorphous silica and quartz; silicas; metal silicates,controlled pore glasses and ceramics; metal oxides and sulfides, orcombinations of these natural or synthetic and organic or inorganicmaterials.

The matrix backbone is preferably selected from agar-agar, agaroses,celluloses, cellulose ethers such as hydroxypropyl cellulose,carboxymethyl cellulose, polyamides such as Poly(meth)acryl-amides,polyvinylalcohols, silicas, and controlled pore glasses.

Especially interesting solid phase materials as matrix backbones aree.g. agar or agarose beads such as Sepharose and Superose beads fromPharmacia Biotech, Sweden and Biogel A from Biorad, USA; dextran basedbeads such as Sephadex, Pharmacia Biotech; cellulose based beads andmembranes such as Perloza cellulose from Secheza, Czechoslovakia;composite beads such as Sephacryl and Superdex, Pharmacia Biotech; beadsof synthetic organic polymers such as Fractogel from Toso-Haas, USA;POROS media from Perceptive Biosystems, USA, Bio-Rex, Bio-Gel P andMacro Prep from Biorad, HEMA and Separon from TESSEK and Hyper D andTrisacryl media from BioSepra, USA, Enzacryl and Azlactone, 3M, USA;beads of siliceous materials such as controlled pore glass, PROSEP, fromBioprocesing, England and Spherocil, BioSepra; and coated silicacomposites in the form of beads or membranes such as ACTI-DISK, ACTI-MODand CycloSep from Arbor Technologies, USA.

Typically, the solid phase matrix backbone, as well as the resultingfunctionalised solid phase matrix may, e.g., be in the form of irregularparticles or spherical beads, membranes or sheets, moulded surfaces, orsticks. The solid phase material may further be fully or partlypermeable or completely impermeable to proteins. In a particularlyinteresting embodiment of the present invention, the matrix is in theform of irregular or spherical beads with sizes in the range of 1-10000μm, preferably 10-1000 μm; such as 10-60 μm for high performanceapplications and such as 50-500 μm, preferably 50-300 μm, forpreparative purposes.

A particular interesting form of matrix is a density controlled matrixin the form of a conglomerate comprising density controlling particles.These conglomerates, which are especially applicable in large scaleoperations for fluidised or expanded bed chromatography as well asdifferent batch-wise chromatography techniques in non-packed columns,e.g. simple batch adsorption in stirred tanks, are described in the WO92/00799, which is hereby incorporated by reference.

The ligands L may be attached to the solid phase material by any type ofcovalent bond known per se to be applicable for this purpose, either bya direct chemical reaction between the ligand and the solid phasematerial or by a preceding activation of the solid phase material or ofthe ligand with a suitable reagent known per se making it possible tolink the matrix backbone and the ligand. Examples of such suitableactivating reagents are epichlorohydrin, epibromohydrin,alyl-glycidylether; bis-epoxides such as butanedioldiglycidylether;halogen-substituted aliphatic compounds such as di-chloro-propanol,divinyl sulfone; carbonyldiimidazole; aldehydes such as glutaricdialdehyde; quinones; cyanogen bromide; periodates such assodium-meta-periodate; carbodiimides; chloro-triazines such as cyanuricchloride; sulfonyl chlorides such as tosyl chlorides and tresylchlorides; N-hydroxy succinimides; 2-fluoro-1-methylpyridiniumtoluene-4-sulfonates; oxazolones; maleimides; pyridyl disulfides; andhydrazides. Among these, the activating reagents leaving a spacer groupSP1 different from a single bond, e.g. epichlorohydrin, epibromohydrin,allyl-glycidylether; bis-epoxides; halogen-substituted aliphaticcompounds; divinyl sulfone; aldehydes; quinones; cyanogen bromide;chloro-triazines; oxazolones; maleimides; pyridyl disulfides; andhydrazides, are preferred.

Especially interesting activating reagents are believed to beepoxy-compounds such as epichlorohydrin, allyl-glycidylether andbutanedioldiglycidylether.

In certain instances the activating reagent may even constitute a partof the functionality contributing to the binding of immunoglobulins tothe solid phase matrix, e.g. in cases where divinyl sulfone is used asthe activating reagent. In other cases the activating reagent isreleased from the matrix during reaction of the functional group withthe matrix. This is the case when carbodiimidazoles and carbodiimidesare used.

The above mentioned possibilities makes it relevant to define thepresence of an optional spacer SP1 linking the matrix M and the ligandL. In the present context the spacer SP1 is to be considered as the partof the activating reagent which forms the link between the matrix andthe ligand. Thus, the spacer SP1 corresponds to the activating reagentsand the coupling reactions involved. In some cases, e.g. when usingcarbodiimides, the activating reagent forms an activated form of thematrix or of the ligand reagent. After coupling no parts of theactivating reagent is left between the ligand and the matrix, and, thus,SP1 is simply a single bond.

In other cases the spacer SP1 is an integral part of the functionalgroup effecting the binding characteristics, i.e. the ligand, and thiswill be especially significant if the spacer SP1 comprises functionallyactive sites or substituents such as thiols, amines, acidic groups,sulfone groups, nitro groups, hydroxy groups, nitrile groups or othergroups able to interact through hydrogen bonding, electrostatic bondingor repulsion, charge transfer or the like.

In still other cases the spacer SP1 may comprise an aromatic orheteroaromatic ring which plays a significant role for the bindingcharacteristics of the solid phase matrix. This would for example be thecase if quinones or chlorotriazines where used as activation agents forthe solid phase matrix or the ligand.

Preferably, the spacer SP1 is a single bond or a biradical derived froman activating reagent selected from epichlorohydrin,allyl-glycidylether, bis-epoxides such as butanedioldiglycidylether,halogen-substituted aliphatic compounds such as 1,3-dichloropropan-2-ol,aldehydes such as glutaric dialdehyde, divinyl sulfone, quinones,cyanogen bromide, chloro-triazines such as cyanuric chloride,2-fluoro-1-methylpyridinium toluene-4-sulfonates, maleimides,oxazolones, and hydrazides.

Preferably the spacer SP1 is selected from short chain aliphaticbiradicals, e.g. of the formula —CH₂—CH(OH)—CH₂— (derived fromepichlorohydrin), —(CH₂)₃—O—CH₂—CH(OH) —CH₂ — (derived fromallyl-glycidylether) or —CH₂—CH(OH)CH₂—O—(CH₂)₄—O—CH₂—CH(OH)—CH₂—derived from butane-dioldiglycidylether; or a single bond.

Due to the risk of leakage of material (e.g. the ligand and/or thespacer) from a solid phase matrix into the eluted product (e.g. theimmunoglobulin) the molecular weight of the ligand (or the ligand+theoptional spacer) is advantageously chosen as low as possible. A majordrawback of using protein A, protein G, synthetic peptides and otherrelatively high molecular weight ligands (e.g. dyes) is that it may bedifficult or even impossible to separate any released ligand (optionallyincluding the spacer) from the eluted immunoglobulin due to the smalldifference between the respective molecular weights and the naturaltendency of the components to bind to each other. This may have adetrimental effect in those cases where the immunoglobulin is to be usedas a therapeutic agent causing allergic chock or other serious symptomsin the patient. The smaller the molecular weight of the ligand(including its spacer) the more efficient can any leaked ligand beseparated from the immunoglobulin product. Another significant advantageof having the smallest possible molecular weight of the ligand (or theligand-spacer arm conjugate) is that any leaked material, which may nothave been separated from the immunoglobulin prior to injection/ingestionin the patient will elucidate a minimum of antigenicity the lower themolecuar weight and therefore in general be relatively more acceptableto the organism than higher molecular weight ligands.

It is therefore, preferred that the ligand L has a molecular weightbelow 500 Dalton, preferably below 400 Dalton, more preferably below 300Dalton, such as below 250 Dalton, or even below 200 Dalton.

With respect to the ligand-spacer arm conjugate (—SP1-L), it ispreferred that the molecular weight is below 500 Dalton, more preferablybelow 400 Dalton, such as below 300 Dalton, or even below 250 Dalton.

According to the invention, the matrix comprises ligands which eitheralone or in combination with a spacer SP1 (and even the matrix backbone)make it possible to bind immunoglobulins thereto. It is found that acrucial part of the ligand is a mono- or bicyclic aromatic orheteroaromatic moiety which may carry one or more substituents, one ofwhich preferably being a substituent comprising an acidic moiety.

The term “mono- or bicyclic” is intended to mean that the core part ofthe moiety in question is consisting of one ring or two fused rings,e.g. as in benzene and naphthalene, respectively, and, thus, not toligands comprising two separate rings as in biphenyl.

It has been found that the structure of the aromatic or heteroaromaticpart of the ligand, L, may cover a very wide spectrum of differentstructures optionally having one or more substituents on the aromatic orheteroaromatic ring(s). However, it seems to be rather decisive whichsubstituents are present on, e.g., a benzene ring as to whether theligand will bind the immunoglobulin(s) efficiently, which is the objectof the present invention, or whether the binding is only moderately orlow.

Even though the ligands are named here and in the following using thenomenclature corresponding to the individual and discrete chemicalcompound, from which they are derived, it should be understood that theactual ligand L is a radical of such a compound.

However, based on our preliminary findings, it is especially preferredto employ matrices comprising aromatic or heteroaromatic groups(radicals) of the following types as functional groups: benzoic acidssuch as 2-aminobenzoic acids, 3-aminobenzoic acids, 4-aminabenzoicacids, 2-mercaptobenzoic acids, 4-amino-2-chlorobenzoic acid,2-amino-5-chlorobenzoic acid, 2-amino-4 -chlorobenzoic acid,4-aminosalicylic acids, 5-aminosalicylic acids, 3,4-diaminobenzoicacids, 3,5-diaminohaenzoic acid, 6-aminoisophthalic acid,4-aminophthalic acid; cinnamic acids such as hydroxyciinamic acids;nicotinic acids such as 2-mercaptonicotink acids; naphthoic acids suchas 2-hydroxy-1-naphthoic acid; quinolines such as 2-meraaptoquinoline;tetrazolacetic acids such as 6-mercapto-1-tetrazolacetic acid;thiadiazols such as 2-mercapto-5-methyl-1,3,4-thiadiazol; benzimidazolssuch as 2-amino-benzimidazol, 2-mercaptobenzimidazol, and2-mercapto-5-nitro-benzimidazol; benzothiazols such as2-aminobenzothiazoL 2-amino-6-nitrobenzothiazol, 2-mercaptobenzothiazoland 2-mercapto-6-ethoxybenzothiazol; benzoxazols such as2-mercaptobenzoxazol; thiophenols such as thiophenol and2-aminothiophenol; 2-(4-aminophenylthio)acetic acid; aromatic orheteroaromatic sulfonic acids and phosphonic acids, such as1-amino-2-naphthol-4-sulfonic acid and phenols such as2-amino-4-nitrophenol. It should be noted that the case where M isagarose, SP1 is derived from vinyl sulfone, and L is 4-amino-benzoicacid is specifically disclaimed in relation to the solid phase matricesaccording to the invention, of. WO 92/16292.

The detailed structure of the ligand seems to determine importantfunctional characteristics relevant for the isolation of immunoglobulinsfrom different sources. Thus, different ligands comprising remote orclosely related aromatic structures seems to result in significantchanges in the binding strength, binding selectivity, binding capacityand overall yield of immunoglobulin when applied in the isolation ofantibodies from different raw materials.

For binding of immunoglobulins at near neutral pH (about pH 5 to pH 9)it is preferred to use a ligand comprising radicals derived from abenzene ring fused with a heteroaromatic ring system, e.g. a ligandselected from benzimidazoles such as 2-mercapto-benzimidazol and2-mercapto-5-nitro-benzimidazol; benzothiazols such as2-amino-6-nitrobenzothiazol, 2-mercaptobenzothiazol and2-mercapto-6-ethoxybenzothiazol; benzoxazols such as2-mercaptobenzoxazol. Not belonging to the former group of ligands butalso preferred for the binding of immunoglobulins at near neutral pH areligands chosen from the group of thiophenols such as thiophenol and2-aminothiophenol.

Thus, as it is clear from the above and the results shown herein, theligand L is preferably selected from radicals having the followingformula-X-A-SUBwherein X designates —O—, —S—, or —NH—, A designates an aromatic orheteroaromatic ring or ring system, and SUB designates one or moresubstituents.

It is understood that X is an integral part of the ligand in that thearomatic or heteroaromatic compound which forms the ligand part of thesolid phase matrix after reaction with an activated matrix backbone,must include a hydroxy group X is —O—), a mercapto group to is —S—) oran amino group (X is —NH—) directly attached the aromatic orheteroaromatic moiety. Examples of such compounds are 3-hydroxy-cinnamicacid, 2-mercapto-benzoic acid, and 2-amino-benzoic acid. It should beunderstood that if the aromatic or heteroaromatic compound comprises,e.g., a hydroxy group as well as an amino group, the resulting solidphase matrix may comprise a mixture of ligand being attached to thelinker through the amino group and through the hydroxy group,respectively.

The aromatic radicals are preferably selected from benzene radicals andnaphthalene radicals

The aromatic radical is preferably a benzene radical such as phenyl,1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2,3-benzenctriyl,1,2,4-benzenetriyl, 1,3,5-benzenetriyl, 1,2,3,4-benzenctetrayl,1,2,3,5-benzenetetrayl, 1,2,4,5-benzenetetrayl, and1,2,3,4,5-benzenepentayl.

The heteroaromatic radicals are preferably selected from monocyclicheteroaromatic radicals such as thiophene, furan, pyran, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, and pyridazine radicals; and bicyclic heteroaromaticradicals such as indole, purine, quinoline, benzofuran, benzimidazole,benzothiazole, and benzoxazole radicals.

The heteroaromatic radical is preferably selected from radicals ofpyridine, benzimidazole, benzothiazole and benzoxazole.

A preferred group of ligands for high purity immunoglobuln isolates ischosen among amino-benzoic acids like 2-amino-benzoic acid,2-mercapto-benzoic acid, 3-aminobenzoic acid, 4-amino-benzoic acid,4-amino-2-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid,2-amino-4-chlorobenzoic acid, 4-aminosalicylic acids, 5-aminosalicylicacids, 3,4-diaminobenzoic acids, 3,5-diaminobenzoic acid,5-aminoisophthalic acid, 4-aminophthalic acid.

Another preferred group of ligands giving a high degree of purity of theisolated immunoglobulin is the group of cinnamic acids such as2-hydroxy-cinnamic acids, 3-hydroxy-cinnamic acid and 4-hydroxy-cinnamicacid.

Still another preferred group of ligands for isolation of high purityimmunoglobulins are derived from the group of heteroaromatic compoundscomprising a carboxylic acid and an amino group as substituents such as2-amino-nicotinic acid, 2-mercapto-nicotinic acid, 6-amino-nicotinicacid and 2-amino-4-hydroxypyrimidine-carboxylic acid.

Agarose matrix backbones and spacers derived from epoxy compounds areespecially relevant in combination with these preferred groups ofligands.

With respect to the substituents on the aromatic or heteroaromaticmoiety, SUB preferably comprises at least one acidic group.

In a particularly interesting embodiment of the present invention, SUBcomprises at least one substituent of the following formula-SP2-ACIDwherein SP2 designate an optional second spacer and ACID designates anacidic group.

In the present context the term “acidic group” is intended to meangroups having a pKa-value of less than about 6.0, such as a carboxylicacid group (—COOH), sulfonic acid group (—SO₂OH), sulfinic acid group(—S(O)OH), phosphinic acid group (—PH(O)(OH)), phosphonic acid monoestergroups (—P(O)(OH)(OR)), and phosphonic acids group (—P(O)(OH)₂). ThepKa-value of the acidic group should preferably be in the range of 1.0to 6.0.

The acidic group is preferably selected from carboxylic acid, sulfonicacid, and phosphonic acid.

The group SP2 is selected from C₁₋₆-alkylene, and C₂₋₆-alkenylene, orSP2 designates a single bond. Examples of relevant biradicals aremethylene, ethylene, propylene, propenylene, iso-propylene, andcyclohexylene. Preferably, SP2 designates methylene, ethylene, or asingle bond.

In one embodiment of the present invention SUB designates one group—SP2-ACID. In this case, SP2 is preferably a single bond.

SUB may, however, designate a substituent —SP2-ACID as well as one ormore further substituent(s) independently selected from hydroxy, amino,cyano, mono- and di(C₁₋₆-alkyl)amino, halogen such as iodo, bromo,chloro, and fluoro, sulfanyl, nitro, C₁₋₆-alkylcarboxy, andaminocarboxy, mono- and di(C₁₋₆-alkyl)aminocarboxy, carboxy, sulfono,sulfonamide, phosphonic ester with C₁₋₆-alkyl, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl optionally substitutedC₁₋₁₂-alkynyl, and optionally substituted C₁₋₁₂-alloxy, thioester, orthe substituent is an oxygen atom which together with two valences of acarbon atom of the aromatic or heteroaromatic moiety form an oxo group.Furthermore, SUB may designate a further group —SP2-ACID as definedabove. It should be understood that the substituents defined for SUBcorrespond to the optional substituents for L.

In another preferred embodiment, SUB designates a substituent —SP2-ACIDas well as one or more further substituent(s) independently selectedfrom hydroxy, amino, cyano, halogen, sulfanyl, nitro, optionallysubstituted C₁₋₆-alkyl methyl, ethyl, propyl, butyl, isobutyl andcyclohexyl, optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkynyl, optionally substituted C₁₋₆-alkoxy, carboxy, and sulfono,or the substituent is an oxygen atom which together with two valences ofa carbon atom of the aromatic or heteroaromatic moiety form an oxogroup. Also in this case, SP2 preferably designates methylene,ethenylene, or a single bond, preferably a single bond.

In the present context, the term “C₁₋₁₂-alkyl” is intended to mean alkylgroups with 1-12 carbon atoms which may be straight or branched orcyclic such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, pentyl, hexyl, octyl, dodecyl, cyclopentyl, cyclohexyl,decalinyl, etc.

The term “optionally substituted C₁₋₁₂-alkyl” is intended to mean aC₁₋₁₂-alkyl group which may be substituted with one or more, preferably1-3, groups selected from carboxy; protected carboxy such as a carboxyester, e.g. C₁₋₆-alkoxycarbonyl; aminocarbonyl; mono- anddi(C₁₋₆-alkyl)-aminocarbonyl; amino-C₁₋₆-alkyl-aminocarbonyl; mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl; amino; mono- anddi(C₁₋₆-alkyl)amino; C₁₋₆-alkylcarbonylamino; hydroxy; protected hydroxysuch as acyloxy, e.g. C₁₋₆-alkanoyloxy; sulfono; C₁₋₆-alkylsulfonyloxy;nitro; phenyl; phenyl-C₁₋₆-alkyl; halogen; nitrilo; and mercapto.

Examples of substituted C₁₋₁₂alkyl groups are carboxy-C₁₋₁₂-alkyl (e.g.carboxymethyl and carboxyethyl), protected carboxy-C₁₋₁₂-alkyl such asesterified carboxy-C₁₋₆-alkyl (e.g. C₁₋₆-alkoxy-carbonyl-C₁₋₁₂-alkylsuch as methoxycarbonylmethyl, ethoxycarbonylmethyl, andmethoxyearbonylethyl), aminocarbonyl-C₁₋₁₂alkyl (e.g. aminocarbonyiethylaminocarbonylethyl and aminocarbonylpropyl),C₁₋₆-alkylaminocarbonyl-C₁₋₁₂-alkyl (e.g. methylaminocarbonylmethyl andethylaminocarbonylmethyl), amino-C₁₋₆-alkyl-aminocarbonyl-C₁₋₁₂-alkyl(e.g. aminomethylaminocarbonylmethyl and aminoethylaminocarbonylmethyl),mono- or di(C₁₋₆-alkyl)amino-C₁₋₆alkylaminocarbonyl-C₁₋₁₂-alkyl (e.g.dimethylaminomethylaminocarbonylmethyl anddimethylaminoethylaminocarbonylmethyl), mono- ordi(C₁₋₆-alkyl)amino-C₁₋₁₂alkyl (e.g. di-methylaminomethyl anddimethylaminoethyl), hydroxy-C₁₋₁₂-alkyl (e.g. hydroxymethyl andhy-droxyethyl), protected hydroxy-C₁₋₁₂-alkyl such asacyloxy-C₁₋₁₂-alkyl (e.g. C₁₋₆-alkanoyloxy-C₁₋₁₂ alkyl such asacetyloxyethyl, acetyloxypropyl, acetyloxybutyl, acetyloxypentyl,propionyloxy -methyl, butyryloxymethyl, and hexanoyloxymethyl).

In the present context, the term “C₂₋₁₂-alkenyl” is intended to meanmono-, di- or polyunsaturated alkyl groups with 2-12 carbon atoms whichmay be straight or branched or cyclic in which the double bond(s) may bepresent anywhere in the chain or the ring(s), for example vinyl,1-propenyl 2-propenyl, hexenyl, decenyl, 1,3-heptadienyl, cyclohexenyletc. Some of the subsituents exist both in a cis and a transconfiguration. The scope of this invention comprises both the cis andtrans forms.

In the present context, the term “C₂₋₁₂-alkynyl” is intended to mean astraight or branched alkyl group with 2-12 carbon atoms andincorporating one or more triple bond(s), e.g. ethynyl, 1-propynyl,2-propynyl, 2-butynyl, 1,6-heptadiynyl, etc.

In the expressions “optionally substituted C₂₋₁₂-alkenyl” and“optionally substituted C₂₋₁₂-alkynyl”, the term “optionallysubstituted” is intended to mean that the moiety may be substituted oneor more times, preferably 1-3 times, with one of the groups definedabove for “optionally substituted C₁₋₁₂-alkyl”.

The term “optionally substituted C₁₋₁₂-alkoxy” designates, as intraditional chemical nomenclature, an optionally substitutedC₁₋₁₂-alkyl-oxy group, which may be substituted one or more times,preferably 1-3 times, with the substituents indicated for “optionallysubstituted alkyl” described above.

The terms “C₁₋₆-alkyl”, “C₂₋₆-alkenyl”, “C₂₋₆-alkynyl”, and“C₁₋₆-alkoxy” reflect the shorter analogues of the “C₂₋₁₂-alkyl”,“C₂₋₁₂-alkenyl”, “C₂₋₁₂-alkynyl”, and “C₁₋₁₂-alkoxy” groups.

The terms “C₁₋₆-alkylene” and “C₂₋₆-alkenylene” is intended to meanbiradicals of the groups defined for “C₁₋₁₂-alkyl” and “C₂₋₆-alkenyl”,respectively.

The present invention should not be bound to any specific theory,however, it is envisaged that the special electronic configuration ofthe aromatic or heteroaromatic moiety in combination with one or moreheteroatoms, which may be located in the heteroaromatic ring system oras a substituent thereon, is involved in the specific binding ofimmunoglobulins, as well as the binding of other proteins.

Thus, in an interesting embodiment of the present invention the ligandcomprises at least one nitrogen, sulfur or phosphorous atom, e.g. as aring atom or as a substituent on the (hetero)aromatic ring, such as anamino or nitro group or a sulfonic acid group or a phosphonic acidgroup.

An especially interesting combination of substituents seem to be anycombination of at least one amino or mercapto group with at least oneacidic group selected firm carboxylic acids, sulfonic acids, andphosphonic acids.

It is envisaged that a combination of two or more of the ligands typedefined herein on the same matrix backbone may lead to certain tocertain advantages with respect to high binding efficiency and/or highpurity of the immunoglobulin.

However, in an important embodiment of the present invention, all of thefunctional groups on the solid phase matrix are substantially identical.

It may also be found to enhance binding efficiency and purity of theproduct by coupling the ligand to a matrix already comprising negativelyor positively charged moieties such as positively charged amino-groupsor negatively charged carboxylic acid, sulfonic acid or phosphonic acidgroups.

The ligand concentration may also be of major significance for thefunctional characteristics of a matrix according to the invention e.g. aligand may show a high degree of selective binding of immunoglobulins atone ligand concentration, while an increase in the ligand concentrationresults in a decrease in the binding selectivity. As is well-known to aperson skilled in the art too high ligand concentrations may lead tostrong binding of unwanted impurities by mechanism of multiple bindingpoints, because the ligands are too closely spaced on the solid phasebackbone. If the ligand concentration is kept low the ligands will bespaced with larger distances and therefore not course the binding ofimpurities by binding at multiple sites.

Another negative effect of too high ligand concentration is the risk ofbinding the wanted protein e.g. the immunoglobulin by multiple bindingsites. Such a multiple binding may lead to difficulties in releasing theprotein e.g. the immunoglobulin with an appropriate elution buffer. Insome instances it may even be necessary to utilise strongly denaturingconditions and/organic solvents for release of the product from such tohighly substituted solid phase matrices—with loss of biological activityas a consequence.

Ligand concentration of solid phase matrices may be disclosed in severaldifferent ways. One way of describing the ligand concentration is todisclose the amount of ligand present per gram of dry matter (e.g. inμmol/g dry matter). This is the result obtained directly if for examplethe ligand concentration is measured by elemental analysis of dried(e.g. freeze-dried) samples of the solid phase matrix. The ligandconcentration may, however, also be disclosed as the amount of ligandpresent on one ml wet and sedimented solid phase matrix (also oftenreferred to as one ml packed bed matrix). This is a figure which iseasily calculated from a determination based on dried solid phase matrix(e.g. μmol/g dry matter), if the dry matter content of the hydratedsolid phase matrix has been determined at the same time (i.e. gram ofdry matter/ml wet sedimented solid phase matrix). Still another way ofdisclosing the ligand concentration is as the amount of ligand presentin one gram of wet, but suction drained matrix. This figure is againeasily calculated from a determination based on dry matter, if the solidphase dry matter content per gram, of wet, but suction drained matrixhas been determined at the same time.

Thus, the ligand concentration of the solid phase matrices of theinvention is preferably in the range of 10-990 μmol/g dry matter ofsolid phase matrix, such as 100-990 μmol/g, more preferably 200-980μmol/g, in particular 250-976 μmol/g;

or

the ligand concentration the solid phase matrices of the invention ispreferably in the range of 1-145 μmol/ml of hydrated, sedimented solidphase matrix, such as 10-120 μmol/ml, more preferably 15-100 μmol/ml, inparticular 20-80 μmol/ml;

or

the ligand concentration the solid phase matrices of the invention ispreferably in the range of 1-130 μmol/g wet, but suction drained solidphase matrix, such as 10-110 μmol/gram, more preferably 20-100 μmol/g,in particular 20-90 μmol/gram.

It is, as should already be clear from the above, the aim of the presentinvention to provide solid phase matrices having a high bindingefficiency.

Thus, the solid phase matrices, which are useful within the scope of thepresent invention must fulfil two of three criteria (a), (b), and (c)(see above), e.g. criteria (a) and (b), criteria (a) and (c), orcriteria (b) and (c). Preferably all three criteria are fulfilled.

With respect to criterion (a), it is highly desirably in combinationwith the other criteria set forth herein or as an alternative thereto,that the solid phase matrix has a binding efficiency of at least 50%when tested at a pH in the range of 2.0 to 10.0 in the “StandardImmunoglobulin Binding Test” described herein. It is envisaged that thebinding efficiency maximum (which can be estimated quite accurately,within half a pH unit, by testing the binding efficiency over an pHrange using, e.g., increments of 0.5 pH units) of most of the matricesaccording to the invention is in the range of 3.0 to 9.0, e.g. in therange 3.0 to 7.0 or in the range of 6.0 to 9.0 depending on the natureof the ligand.

It has been found that the binding efficiency at pH 4.5 and pH 7.0 isespecially relevant when performing a general evaluation of a solidphase matrix for isolation of immunoglobulins, thus, in a preferredembodiment, the present invention relates to a solid phase matrix havinga binding efficiency of at least 50% at pH 4.5 or pH 7.0, in the“Standard Immunoglobulin Binding Test” described herein.

Thus, in a particularly interesting embodiment of the present invention,the solid phase matrix has a binding efficiency of at least 50,preferably at least 60%, more preferably at least 70%, in particular atleast 80%, such as at least 90%, in the “Standard Immunoglobulin BindingTest” described herein, at least one pH-value of the solvent in therange of pH 1.0 to pH 11.0, in particular in the range of pH 3.0 to pH9.0, and more particularly at pH 4.5 or 7.0.

Furthermore, it is also the aim of the present invention to providesolid phase matrices which are able to bind a vide range ofimmunoglobulins, so that the end user can rely on one solid phase matrixinstead of an number of products which has to be tested individually foreach clone of immunoglobulins.

Thus, with respect to the criterion (b), the solid phase matrixpreferably has an average binding efficiency of at least 50%, such as atleast 60%, preferably at least 70%, especially at least 80%, inparticular at least 90%, for the immunoglobulins tested in the“Monoclonal Antibody Array Binding Tests” when tested at a pH in therange of 2.0 to 10.0, such as in the range of 3.0 to 9.0, e.g. in therange of 3.0 to 7.0 or in the range of 6.0 to 9.0. Typically, thebinding efficiency is determined at two pH values, e.g. at pH 4.5 and pH7.0, and the optimum is then found by varying the pH value in incrementsof 0.5 around the one of the two pH values giving the most promisingbinding efficiency.

The functional stability of the matrix, which is interesting andimportant with respect to lower risk of leaching and the possibility ofregeneration, may be influenced by the chemical structure of the ligand,i.e. the stability to harsh regeneration conditions such as 1 M sodiumhydroxide is dependent on the ligand structure, as well as the matrixbackbone and any spacer moiety.

Therefore, with respect to criterion (c), it is a preferred that thestability (see example 8) of the solid phase matrix in 1 M NaOH is sothat incubation of the matrix in 1 M NaOH in the dark at roomtemperature for 7 days reduces the binding efficiency at a pH in therange of one pH unit lower than the binding maximum pH to one pH unithigher than the binding maximum pH value, as determined according to the“Standard Immunoglobulin Binding test” described herein, with less than50%, preferably less than 25%, compared to a corresponding untreatedmatrix. Preferably the reduction is less than 15%, such as at less than10%, in particular less than 5%.

It has been found that solid phase matrices comprising a functionalisedmatrix backbone carrying a plurality of functional groups of thefollowing formulaM-SP1-X-A-SP2-ACID

-   -   wherein M designates the matrix backbone; SP1 designates a        spacer; X designates —O—, —S—, or —NH—; A designates a mono- or        bicyclic optionally substituted aromatic or heteroaromatic        moiety; SP2 designates an optional spacer; and ACID designates        an acidic group;        with the first proviso that the molecular weight of the ligand        -L is at the most 500 Dalton,        are novel in themselves (specifically disclaiming 4-aminobenzoic        acid disclosed in K. L. Knudsen et al, Analytical Biochemistry,        vol. 201, p. 170, 1992 and WO 92/16292, which has been used for        the isolation of immunoglobulins in combination with lyotropic        salts, as a ligand), and that they are well suited for the        isolation and/or purification of immunoglobulins as well as for        the isolation and/or purification of proteins and other        biomolecules in general.

It has furthermore been found that the above-mentioned solid phasematrices comprising functional groups of the formula M-SP1-X-A-SP2-ACID,are equally applicable for pH-dependent reversible binding of proteinsand other biomolecules.

Thus, the present invention also provides a method for the isolation ofproteins from a solution containing one or more of proteins, comprisingthe following operations:

-   a) contacting a solution containing one or more proteins having a pH    in the range of 1.0 to 6.0 and a total salt content corresponding to    a ionic strength of at the most 2.0 with a solid phase of the    formula M-SP1-X-A-SP2-ACID, whereby at least a part of the proteins    becomes bound to the solid phase matrix;-   b) separating the solid phase matrix having proteins bound thereto    from the solution;-   c) optionally washing the solid phase matrix; and-   d) contacting the solid phase matrix with an eluent in order to    liberate one or more of the proteins from the solid phase matrix,    wherein the eluent used comprises less than 10% (v/v) of organic    solvents.

The pH of the solution containing the proteins is preferably in therange of 1.0 to 6.0, such as 2.0 to 6.0, especially in the range of 3.0to 5.5, such as 4.0 to 5.0, and the pH of the eluent is in the range of6.0 to 11, preferably in the range of 6.0 to 9.0.

As in the method for the isolation of the immunoglobulins, the totalsalt content of the solution containing the proteins preferablycorresponds to a ionic strength of at the most 20, such as in the rangeof 0.05 to 2.0, in particular in the range of 0.05 to 1.4, especially inthe range of 0.05 to 1.0, and/or the concentration of lyotropic saltspreferably is at the most 0.4 M, such as at the most 0.3 M, inparticular at the most 0.2 M, especially at the most 0.1 M. Furthermore,as above, it is advantageous to use a negatively charged detergent inthe contacting step (operation (a)) and/or in the washing step(operation (c)). Preferably, the washing step (operation (c)) preferablyimplies the use of an inorganic or organic salt buffer comprising anegatively charged detergent.

The method for the isolation of proteins and other biomolecules may beemployed for a number of proteins, examples of which are proteases suchas pro-enzymes, trypsins, chymotrypsins, subtilisin, pepsin,plasminogen, papain, renin, thrombin, and elastase; lipases,glucosidases; xylanases; lectins; albumins; proteins from fermentationsbroths; protein from milk and whey; proteins from blood, plasma, andserum; proteins from fish waste; proteins from slaughterhouse waste suchas organ and tissue extracts, e.g. alkaline phosphatase from bovineintestines; and proteins from vegetable extracts such as potato, tomato,coconut, e.g. horse radish peroxidase.

Synthesis of Solid Phase Matrices

Generally a solid phase matrix may be derivatised so as to comprisecovalently linked ligands according to the invention be methods know perse, e.g., activation of the solid phase matrix with a suitable reagentknown per se followed by coupling of the ligand to the activated matrix,optionally incorporating a spacer SP1 between the ligand and the matrixby coupling the spacer to the activated matrix first followed bycoupling the ligand to the spacer via a suitable condensation reagent oreven a second activation of the spacer followed by coupling of theligand.

The sequence and choice of reagents may depend on the actual ligand tobe coupled and the solid phase material to be derivatised withconsideration to, e.g., the content of reactive groups such as hydroxyl,amino, mercapto, and silanols etc. In some cases it may be preferable toactivate or derivatise the ligand instead of the solid phase matrixfollowed by a reaction of the derivatised ligand with the solid phasematrix.

Thus, in a preferred method for of synthesising a solid phase matrixaccording to the invention, the solid phase matrix is first reacted witha reagent able to react with the solid phase matrix and thereby activateit for further reaction with the ligand, optionally washing away theactivation reagent followed by a reaction of the activated solid phasematrix with a solution comprising the ligand and optionally followed bywashing the solid phase matrix comprising the covalently immobilisedligand with one or more suitable solutions cleaning the matrix forsurplus reactants.

In some cases in may be possible to combine the activation and thecoupling of the ligand by mixing the two reagents and let the reactionstake place in parallel. This is a great advantage as it saves costs andtime as well as minimising the of waste water. Thus, the activation andthe coupling step is preferably performed in one combined step.

Furthermore, it is a significant advantage if the activation and/or thecoupling reaction can be performed without the need to add organicsolvents to the reaction medium. These organic solvents are often usedto solubilise the reactive reagents or to ensure that hydrolysis ofreactive species are kept at a minimum. However, the use of organicsolvents adds to the cost and risk of the process because of the risk ofexplosions, the risk of health damage, the waste problems and therelatively high cost of the solvents themselves. Thus, the activationand/or the coupling procedure is preferably performed without theaddition of any organic solvent to the reaction medium.

EXAMPLES

The invention is illustrated by the following examples 1-15:

1. Derivatisation of Solid Phases

1A) Epichlorohydrin Activation of Agarose Beads

Activation of Agarose Beads from Hispanagar:

“High” Level of Activation:

Approximately 1000 ml of a 1:1 suspension of agarose beads in water(Hispanagar, 6% agarose beads, particle size 100-140 μm) was washed withwater on a sintered glass funnel followed by suction draining for oneminute. 700 gram of wet, but drained agarose beads were weighed into amixture of 560 ml water and 70 ml 32,5% w/v sodium hydroxide. Thissuspension were then added 90 ml epichlorohydrin (ALDRICH cat. no.:E105-5) followed by gentle stirring with a paddle at room temperature(20-25° C.) for 6 hours. The agarose beads were then washed on a suctionfilter with approx. 20 litres of water and finally suspended in water.The activated agarose beads were found to be stable in this suspensionfor several weeks when stored at 4° C.

The concentration of active epoxy groups on the activated agarose beadswere determined by thiosulfate titration as described in Porath, J.,Låås, T., Janson, J.-C. Journal of Chromatography, vol. 103, pp. 49-69,1976 and Sundberg, L, & Porath, J., Journal of Chromatography, vol 90,pp 87-98, 1974. The results from this titration indicated that theactivated beads had a concentration of 70 μmol epoxy-groups per gram ofwet, but suction drained beads, corresponding to 972 μmol/g drymatter,or 54 μmol/ml wet sedimented beads (aqueous solution).

“Low” Level of Activation:

For production of a matrix with a lower content of active epoxy groupsthe same procedure as described above was followed with the exemptionthat the reaction mixture consisted of: 200 g wet, but suction drainedagarose beads, 160 ml water, 20 ml 2 M sodium hydroxide and 11.5 mlepichlorohydrin.

Thiosulfate titration indicated the presence of 21 μmol epoxy groups pergram wet, but suction drained beads, corresponding to 292 μmol/g drymatter, or 16 μmol/ml wet sedimented beads (aqueous solution).

Actuation of Agarose Beads from Pharmacia and Biorad;

The same activation procedure as described above were employed for theactivation of agarose beads from Pharmacia (Sepharose 4B and Sepharose6B) and Biorad (Biogel A-5m Gel, particle size 38-75 μm and Biogel A-15mGel particle size 75-150 μm).

Titration of active epoxy groups on these solid phases gave thefollowing results:

-   -   μmol epoxy groups per gram drained beads:

Sepharose 4B: 40 Sepharose 6B: 52 Biogel A5m Gel: 65 Biogel A15m Gel: 461B) Epichlorohydrin Activation of Fractogel

Fractogel TSK HW-55 (F), particle size 32-63 μm, from MERCK (cat. no.:14981) and Fractogel TSK HW-65 (F), particle size 32-63 μm, MERCK (cat.no.: 14984) were activated with epichlorohydrin with the same procedureas described above for agarose beads. The resulting concentration ofactive epoxy groups on these solid phases were 98 and 53 μmol/g ofdrained beads respectively.

1C) Butanedioldiglycidyl Ether Activation of Agarose Beads

100 gram 6% agarose beads from Hispanagar was washed with water on asintered glass funnel and drained by suction for one minute. The beadswere then suspended in 75 ml 0.6 M NaOH and hereafter added 75 ml1,4-butanediol diglycidyl ether. Gentle stirring with a paddle wasperformed at room temperature for 18 hours whereafter the matrix waswashed with water approx. 3 litre).

Thiosulfate titration of the amount of epoxy groups incorporated intothe matrix gave a content of 55 μmol/g suction drained matrix.

1D) Divinyl Sulfone Activation of Agarose Beads.

Activation of Agarose Beads from Hispanagar:

Approximately 1400 ml of a 1:1 suspension of agarose beads in water(Hispanagar, 6% agarose beads, particle size 100-140 μm) was washed withdemineralised water on a sintered glass funnel followed by suctiondraining for one minute. 700 gram of wet, but drained, agarose beadswere weighed into 350 ml 0.5 M potassium phosphate buffer pH 11.5. 35 mldivinyl sulfone was added and the resulting suspension was paddlestirred at room temperature for 2 hours. The matrix was then transferredto a sintered glass funnel and washed with 20 litres of water, 5 litresof 30% ethanol in water and finally 5 litres of water. The resultingactivated matrix was determined to have a content of 45 μmol activevinyl groups per gram suction drained beads as determined by thethiosulfate titration method.

1E) Coupling of Ligands to Activated Matrices

General Coupling Procedure:

All couplings of different ligands to the activated matrices mentionedin example 1A-D were performed according to the following generalprocedure:

-   1) The activated beads were washed on a suction filter with 2-3    volumes of demineralised water. The beads were drained by slight    suction on a sintered glass funnel and 20 g of wet, but drained gel    were weighed into a 100 ml plastic bottle with screw cap.-   2) 1 g of ligand was dissolved in 20 ml of water and titrated to pH    10.5-11.0 with 2 M sodium hydroxide (for some ligands with low    solubility the pH was adjusted to pH 11.5-12.5). The resulting    solution was mixed with the activated matrix. The gel was incubated    with the solution by gentle mixing on a roller mixer for 18 hours at    room temperature.-   3) The gel was then washed with 2 litres of water.

In those instances where the ligand had poor solubility in water, a 50%ethanol solution was employed for dissolution instead followed bytitration to pH 10.5-11.0 with 2 M sodium hydroxide. At the sameinstances the final washing with water was substituted with one washingstep of 1 litre 50% ethanol followed by another washing step with 1litre of water.

When divinyl sulfone activated agarose was used for coupling the pH ofthe coupling mixture was adjusted to pH 11.5 instead of 12.6.

Whenever possible the concentration of coupled ligand on the matriceswas determined by elementary analysis of Carbon, Hydrogen, Nitrogen,Oxygen and Sulfur. In some instances it was furthermore possible todetermined the amount of coupled ligand by acid-base titration ofcharacteristic functional groups on the coupled ligand.

Coupling of Ligands to Epoxy-Activated 6% Agarose Beads:

The following chemical substances (ligands) were coupled toepichlorohydrin activated 6% agarose beads (Example 1-A) (Hispanagar,particle size 100-140 μm) according to the above given general couplingprocedure:

2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid,2,5-dihydroxybenzoic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamicacid, 4-hydroxycinnamic acid, 3,5-dinitrosalicylic acid,2-hydroxy-3-methoxybenzoic acid, 3-hydroxy-4-methoxybenzoic acid,2-hydroxy-5-met-hoxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid,3,5-dimethoxy-4-hydroxybenzoic acid, 2-amino-4,5-dimethoxybenzoic acid,5-sulfosalicylic acid, 5-chlorosalicylic acid,4-hydroxy-3,5-di-nitrobenzoic acid, 2-aminobenzoic acid, 3-aminobenzoicacid, 4-aminobenzoic acid, 2-amino-3,5 diiodobenzoic acid,2-mercaptobenzoic acid, 2-mercaptonicotinic acid, aniline-2-sulfonicacid, 2-pyridylhydroxymethanesulfonic acid, 4-acetamidophenol,5-mercapto-1-tetrazolacetic acid, 1-hydroxy-2-naphthoic acid,3-hydroxy-2-naphhtoic acid, 2-hydroxy-1-naphthoic acid,2,3-pyridine-dirboxylic acid, 4-pyridylthioacetic acid,2-pyrimidylthioacetic acid, 2-mercaptochinohine, imidazole,2-mercaptoimidazole, 2-mercapto-1-methylimidazole,3-mercapto-1,2,4-trazole, 5-merapto-1-methyltetrazole,2-mercaptothiazoline, 2-mermapto-1-methyl-1,3,4-tidiaile,2,5-di-mercapto-1,3,4-thiadiazole, benzimidazole,2-hydroxybenzimidazole, 2-aminobenzidazole, 2-mercaptobenzimidazole,2-mercapto-5-nitrobenzimidazole, benzotiazole, 2-ammobenzothiazole,2-amino-6-nitro-benzothiazole, 2-amino-4-ethoxybenzothiazole,2-mercaptobenzthiole, 6-ethoxy-2-mercaptobenzothiamle,6-amino-2,5-dihydroiraidazol-2,1-b)benzothiazde, 2-mercaptobenzoxazole,2-(2-hydroxyphenyl)benzoxazole, phenol, 2-chlorophenol, 3-chlorophenol,4-chlorophenol, 2,4,6-trimethylphenol, 2,3,5-trimethylphenol,4-methoxyphenol, 2,5-dimethoxyphenol, 3,4,5-trimethoxyphenol,thiophenol, 4-chlorothiophenol, 2-aminothiophenol, benzyl mercaptan,4-methoxybenzyl mercaptan, 4-methylthio-m-cresol, aniline,2,4-dimethylani-line, 3,5-dimethoxyaniline, 3,4,5-trimethoxyaniline,2-methylmercaptoaniline, 4-methylmercap-toaniline,2,4,6-tri-methyl-m-phenylendiamine, 2,3-dicyanhydrochinone,2-phenylphenol, 4-phenylphenol, 4-benzyloxyphenol,4,4-diaminophenylsulfone, 2-hydroxypyridie, 2,3-di-hydroxypyridine,2,6-dihydroxypyridine, 2-hydroxy-5-nitropyridine,3-cyano-4,6-dimethyl-2-hydroxypyridine, 4-hydroxy-2-mercaptopyridine,2-mercaptopyridine, 2-aminopyridine, 4-amino-2-chlorobenzoic acid,3-amino-4-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid,2-amino-4-chlorobenzoic acid, 2-amino-5-nitrobenzoic acid,4-aminosalicylic acid, 5-aminosalicylic acid, 3,4 diaminobenzoic acid,3,5-diaminobenzoic acid, 4-aminomethylbenzoic acid, 5-aminoisophthalicacid, 4-aminophthalic acid, 4-aminohippuric acid,3-amino-1,2,4-triazole-5-carboxylic acid, 1-amino-2-naphthol-4-sulfonicacid, 2-(4-aminophenylthio) acetic acid, 2-amino -4-nitrophenol,4-aminophenylacetic acid, 1-aminocyclohexanecarboxylic acid,2-aminobenzylalcohol.

The ligand concentration as determined by elementary analysis onfreeze-dried samples on the respective matrices generally all were inthe range between 50 to 70 μmol of ligand per gram of wet, but suctiondrained matrix.

1F) Coupling of Ligands to Various Solid Phases

The following other solid phases: epoxy activated agarose beads fromHispanagar, activated to a low level (Example 1-A), expoxy activatedagarose beads from Pharmacia and Biorad (Example 1-A), epoxy activatedFractogel from Merck (Example 1-B), butanedioldiglycidyl ether activatedagarose beads from Hispanagar (Example 1-C), and divinyl sulfoneactivated agarose beads from Hispanagar (Example 1-D) were each coupledwith 2-mercapto-benzoic acid (2-MBA), 4-amino-benzoic acid (4-ABA), and2-mercapto-benzimidazole (2-MBI).

The general coupling procedure described above in Example 1-E wasfollowed during all couplings.

The obtained ligand concentrations were determined by elemental analysison freeze-dried samples of the respective matrices and is calculated andgiven as μmol of ligand per gram of wet, but suction dried matrix (onegram of wet, but suction dried matrix corresponds to approx. 1.1-1.3 mlsedimented beads, while the dry matter content may vary considerablymore between the different type of beads.

Ligand Concentrate for the Synthesised Solid Phase Matrices:

(Stated as μmol/g Wet, but Suction Drained Beads)

Epoxy activated agarose beads from Hispanagar, activated to a low level(Example 1-A):

2-MBA 4-ABA 2-MBI 18 20 20

Epoxy activated agarose beads from Pharmacia and Biorad (Example 1-A).:

Matrix\Ligand 2-MBA 4-ABA 2-MBI Sepharose 4B 37 40 38 Sepharose 6B 51 4750 Biogel A5m 59 60 58 Biogel A15m 44 43 41

Epoxy activated Fractogel from Merck (Example 1-B):

Matrix\Ligand 2-MBA 4-ABA 2-MBI Fractogel TSK HW-55 96 92 98 FractogelTSK HW-65 53 51 53

Butanedioldiglycidyl ether activated agarose beads from Hispanagar(Example 1-C):

2-MBA 4-ABA 2-MBI 51 48 44

Diyinyl sulfone activated agarose beads from Hispanagar (Example 1-D):

2-MBA 4-ABA 2-MBI 45 45 422. Standard Immunoglobulin Binding Test

For the purpose of testing all the different solid phase materialssynthesised according to example 1 a standardised test, which can bereproduced any time, has been devised. The test is designed to determinethe immunoglobulin binding efficiency of the different matrices understandardised conditions with respect to composition and pH of the rawmaterial.

To ensure maximal relevancy of the test for isolation of monoclonalantibodies from dilute cell culture supernatants we have simulated theconditions used for culturing hybridoma cells by mixing a typical cellculture media with fetal calf serum and added purified mouseimmunoglobulin to this “artificial culture supernatant.” All reagentsare standard reagents and commercially available.

Definition of “Artificial Culture Supernatant”:

For 250 ml solution:

-   236.5 ml cell culture growth medium, DMEM (Imperial, UK, cat. no.:    7-385-14)-   12.5 ml fetal calf serum (Life Technologies, Denmark, cat. no.:    10106-060)-   1,0 ml purified polyclonal murine IgG (Sigma, USA, cat. no.: 1-8765,    10 mg/ml)-   0.244 g sodium azide (Sigma, USA, cat. no.: S-2002),    resulting in a solution containing: 40 μg murine IgG/ml, 5% fetal    calf serum, and 15 mM sodium azide, and having a pH of approx. 8.0.

This solution was shown to be stable at 4° C. for several weeks withoutany deterioration of the immunoglobulins.

Standard Procedure

-   1) Approximately 100 mg of the matrix to be tested is washed with 10    ml demineralised water on a sintered glass funnel followed by    suction draining for 60 seconds. 100 mg of wet (drained) solid phase    matrix is weighed into a 3.0 ml test tube and 2.50 ml “artificial    culture supernatant” having the pH value at which the matrix is to    be tested is added. The test tube is closed with a stopper, and the    suspension is incubated on a roller mixer for 2 hours at room    temperature (20-25° C.) The test tube is then centrifuged for 5 min.    at 2000 RPM in order to sediment the matrix. The supernatant is then    isolated from the solid-phase matrix by pipetting into a separate    test tube, avoiding the carryover of any matrix particles. Following    this a determination of the concentration of non-bound    immunoglobulin in the supernatant is performed by single radial    immunodiffusion (as described in D. Catty and C Raykundalia    “Antibodies—a practical approach” Vol I, pp. 137-68, 1988) using    rabbit anti mouse immunoglobulins as the precipitating antibody    (DAKO, Denmark, cat. no.: Z109).

The percentage of mouse immunoglobulin bound to the matrix is thencalculated according to the following formula:Percentage bound=(1−(conc. supernatant/conc. starting material))×100%

The precision of this method is better than +/−5%.

2A) Screening for High Immunoglobulin Binding Efficiency

The above described standard procedure for testing the bindingefficiency was used for testing a broad range of different solid phasematrices based on epichlorohydrin activated 6% agarose beads fromHispanagar and synthesised according to example 1A and 1E.

The results of the binding test performed at pH 4.5 and pH 7.0respectively is presented in the Table I below:

TABLE I Capacity at Capacity at Ligand pH 4.5 pH 7.0 2-hydroxybenzoicacid 0 0 3-hydroxybenzoic acid 0 30 4-hydroxybenzoic acid 0 02,5-dihydroxybenzoic acid 60 0 2-hydroxycinnamic acid 20 03-hydroxycinnamic acid 80 0 4-hydroxycinnamic acid 40 03,5-dinitrosalicylic acid 0 0 2-hydroxy-3-methoxybenzoic acid 0 03-hydroxy-4-methoxybenzoic acid 40 0 2-hydroxy-5-methoxybenzoic acid 0 04-hydroxy-3-methoxybenzoic acid 0 0 3,5-dimethoxy-4-hydroxybenzoic acid0 30 2-amino-4,5-dimethoxybenzoic acid 20 0 5-sulfosalicylic acid 0 05-chlorosalicylic acid 0 0 4-hydroxy-3,5-dinitrobenzoic acid 0 02-aminobenzoic acid 80 0 3-aminobenzoic acid 100 0 4-aminobenzoic acid90 0 2-amino-3,5-diiodobenzoic acid 0 0 2-mercaptobenzoic acid 100 02-mercaptonicotinic acid 100 0 aniline-2-sulfonic acid 0 02-pyridylhydroxymethansulfonic acid 0 0 4-acetamidophenol 0 05-mercapto-1-tetrazole acetic acid 70 0 1-hydroxy-2-naphthoic acid 0 03-hydroxy-2-naphthoic acid 0 0 2-hydroxy-1-naphthoic acid 60 0pyridine-2,3-dicarboxylic acid 0 0 4-pyridylthioacetic acid 0 02-pyrimidylthioacetic acid 0 0 2-mercaptochinoline 80 60 imidazole 0 02-mercaptoimidazole 0 0 2-mercapto-1-methylimidazole 20 03-mercapto-1,2,4-triazole 0 0 5-mercapto-1-methyltetrazole 0 02-mercaptothiazoline 20 0 2-mercapto-5-methyl-1,3,4-thiadiazole 0 202,5-dimercapto-1,3,4-thiadiazole 100 20 benzimidazole 0 02-hydroxybenzimidazole 0 0 2-aminobenzimidazole 40 202-mercaptobenzimidazole 70 70 2-mercapto-5-nitrobenzimidazole 80 90benzothiazole 0 0 2-aminobenzothiazole 20 02-amino-6-nitro-benzothiazole 80 60 2-amino-6-ethoxy-benzothiazole 0 02-mercaptobenzothiazole 70 60 6-ethoxy-2-mercaptobenzothiazole 20 406-amino-2,5-dihydroimidazo(2,1- 0 20 b)benzothiazole2-mercaptobenzoxazole 80 60 2-(2-hydroxyphenyl)benzoxazole 0 0 phenol 00 2-chlorophenol 0 0 3-chlorophenol 0 0 4-chlorophenol 0 202,4,6-trimethylphenol 20 0 2,3,5-trimethylphenol 20 202,6-dimethoxyphenol 0 0 3,4,5-trimethoxyphenol 0 0 thiophenol 70 604-chlorothiophenol 100 70 2-aminothiophenol 70 50 benzyl mercaptan 0 0aniline 20 20 2,4-dimethylaniline 0 0 3,4,5-trimethoxyaniline 0 02-methylmercaptoaniline 60 0 2,4,6-tri-methyl-m-phenylendiamine 20 02,3-dicyanhydrochinone 20 0 2-phenylphenol 0 0 4-phenylphenol 20 204-benzyloxyphenol 0 0 1,4-diaminophenylsulfone 20 0 2-hydroxypyridine 00 2,3-dihydroxypyridine 20 0 4-hydroxy-2-mercaptopyridine 60 404-amino-2-chlorobenzoic acid 0 40 3-amino-4-chlorobenzoic acid 0 02-amino-5-chlorobenzoic acid 80 0 2-amino-4-chlorobenzoic acid 40 02-amino-5-nitrobenzoic acid 0 0 4-aminosalicylic acid 80 205-aminosalicylic acid 80 30 3,4-diaminobenzoic acid 80 03,5-diaminobenzoic acid 60 0 4-aminomethylbenzoic acid 0 05-aminoisophthalic acid 60 20 4-aminophthalic acid 60 20 4-aminohippuricacid 0 20 3-amino-1,2,4-triazol-5-carboxylic 0 20 acid1-amino-2-naphthol-4-sulfonic acid 80 20 2-(4-aminophenylthio)aceticacid 80 0 2-amino-4-nitrophenol 80 20 4-aminophenylacetic acid 0 01-aminocyclohexancarboxylic acid 0 0 (reference) 2-aminobenzylalcohol 200

As can be seen from the table some ligands do not bind theimmunoglobulin at all while others show very efficient binding in therange of 80-100% and still other ligands show intermediate bindingefficiencies in the range of 30-60%.

As can be seen from the result from 1-aminocyclohexancarboxylic acid(reference), an aromatic or heteroaromatic moiety seems to be requiredfor efficient binding.

3. Monoclonal Antibody Array Binding Test

The following example illustrates the differences in binding efficiencybetween prior art solid phase matrices and solid phase matricesaccording to the invention for immunoglobulin purification.

For the comparative study 7 different cell lines capable of producing 7different monoclonal antibodies were acquired from the American TypeCulture Collection (ATCC) and propagated according to a standardprocedure as described below. Hereafter the binding efficiency of eachmonoclonal antibody was tested with each of the solid phases: protein Aagarose (prior art matrix), Avidchrom (prior art matrix) andepoxy-linked 2-mercapto-benzoic acid agarose, 4 -amino-benzoic acidagarose and 2-mercapto-benzimidazole agarose.

The study was designed to determine the antibody binding efficiencyduring batch incubation of the 5 different solid phases with culturesupernatants from the 7 different commercially available cell lines.

Monoclonal Antibodies

Cell lines: The following seven cell lines available from the AmericanType Culture Collection were included in the standardised set-up:

ATCC cat no. Immunoglobulin type produced HB 134 Mouse IgG₁ HB 8279Mouse IgG_(2b) HB 8445 Mouse IgG₃ CRL 1852 Mouse IgG₁ HB 121 MouseIgG_(2a) HB 8857 Rat IgG₁ CRL 8018 Mouse IgM

Cell line HB-8279 was redeposited with th American Type CultureCollection P.O. Box 1549, Manassas, Va. USA, 20108 on Jun. 22, 2007, andaccorded ATCC Accession No. SD-5662.

Cultures: The monoclonal antibody culture supernatants used in the studywere produced by culture of the corresponding mouse and rat hybridomacells in a medium containing fetal calf serum (RPMI-X, Medicult, Denmarkcat. no. 20230500+5% fetal calf serum, Imperial, United Kingdom, cat.no. 83041). The methodology used for culturing the five cell lines iswell established in the prior art and described in G. Brown and N. R.Ling “Antibodies—a practcal approach” Vol I, pp. 81-104, 1988). After 3weeks of culture the cells were removed by centrifugation and thesupernatant filtered to remove any remaining particles. Theconcentration of monoclonal antibody in the five different culturesupernatants were determined by single radial immunodiffusion (asdescribed in D. Catty and C Raykundalia “Antibodies—a practicalapproach” Vol I, pp. 137-168, 1988) using rabbit anti mouseimmunoglobulins and rabbit anti rat immunoglobulins as the precipitatingantibodies (DAKO, Denmark, cat. no.: Z109 and Z147) and found to be inthe range of 30 to 60 μg/ml for all clones. Hereafter the content ofmonoclonal antibody in each culture supernatant was standardised bydilution to reach a final concentration of 30 μg/ml. To ensure similarconditions for all the supernatants the dilution was performed withculture medium including 5% fetal calf serum.

Solid phases: Protein A agarose from Repligen Corporation, USA, cat.no.: IPA-300. lot no.: RN 2917; Avidchrom from Unisyn Technologies, USA,cat. no.: 3100-0025, lot no.: 96-0404-1; 2-mercapto-benzoic acidagarose, 4-amino-benzoic acid agarose and 2-mercapto-benzimidazoleagarose were based on epichlorohydrin activated 6% agarose beads fromHispanagar, Spain and synthesised as described in example 1A and 1E. Theligand concentrations were measured by elemental analysis and found tobe 65, 69 and 69 μmoles/gram wet, but drained matrix respectively(corresponding to 903, 958 and 958 μmoles/g dry matter as measured byelemental analysis on freeze dried samples).

The five different solid phase matrices were tested for their monoclonalantibody binding efficiency by incubating them with the 7 differentmonoclonal antibody supernatants (standardised at 30 μg antibody/ml)according to the following procedure:

Standard Procedure for the “Monoclonal Antibody Array Binds Test”:

Approximately 100 mg of the matrix to be tested is washed with 10 mldemineralised water on a sintered glass funnel followed by suctiondraining for 60 seconds. 100 mg of wet (drained) solid phase matrix isweighed into a 3.0 ml test tube and 4.0 ml monoclonal antibody culturesupernatant adjusted to the pH value at which the matrix is to be testedis added. The test tube is closed with a stopper, and the suspension isincubated on a roller mixer for 2 hours at room temperature (20-25° C.).The test tube is then centrifuged for 5 min. at 2000 RPM in order tosediment the matrix. The supernatant is then isolated from the solidphase matrix by pipetting into a separate test tube, avoiding thecarry-over of any matrix particles. Following this a determination ofthe concentration of non-bound immunoglobulin in the supernatant isperformed by single radial immunodiffusion.

The percentage of monoclonal antibody bound to the matrix is thencalculated according to the following formula:Percentage bound=(1−(conc. in supernatant/30 μg/ml))×100%

The precision of this method is better than +/−5%.

pH Adjustments to Culture Supernatants for The Different Solid Phases:

Protein A agarose: The monoclonal antibody culture supernatants wereadjusted to pH 8.2 by the addition TRIS/HCl to a final TRISconcentration of 0.05 M.

Avidchrom: The monoclonal antibody culture supernatants were adjusted topH 7.4 by addition of potassium hydrogen phosphate/HCl to a finalphosphate concentration of 0.05 M.

2-mercapto-benzoic acid and 4-amino-benzoic acid agarose: The monoclonalantibody culture supernatants were adjusted to pH 4.5 by addition ofacetic acid/sodium hydroxide to a final acetic acid concentration of0.05 M.

2-mercapto-benzimidazole The monoclonal antibody culture supernatantswere adjusted to pH 7.0 by addition of potassium hydrogen phosphate/HClto a final phosphate concentration of 0.05 M.

Binding efficiency % Clone Adsorbent ATCC cat. no. Protein Subtype 2-MBA4-ABA 2-MBI A Avidchrom HB 134 100 100 75 0 50 Mouse IgG₁ HB 8445 100100 95 80 80 Mouse IgG₃ CRL 1852 100 100 55 20 40 Mouse IgG₁ HB 8279 9595 75 70 50 Mouse IgG_(2b) HB 121 90 100 85 100 40 Mouse IgG_(2a) HB8857 95 100 95 95 100 Rat IgG₁ CRL 8018 85 60 45 0 10 Mouse IgM Averagebinding 95 94 75 52 53 efficiency % 2-MBA: 2-mercapto-benzoic acidagarose (epichlorohydrin) 4-ABA: 4-amino-benzoic acid agarose(epichlorohydrin) 2-MBI: 2-mercapto-benzimidazole agarose(epichlorohydrin)

As can be seen from the table the solid phase matrices according to theinvention i.e. 2-mercapto-benzoic acid agarose, 4-amino-benzoic acidagarose and 2-mercapto-benzimidazole agarose exhibits a very constanthigh binding efficiency with the different clones (typically in therange of 50-100% binding), while the prior art solid phase matrices,protein A agarose and Avidchrom, exhibits much more varying bindingefficiency (in the range from 0-100% binding). The average bindingefficiency has been calculated for each adsorbent and it is also fromthese data seen that the prior art adsorbents with average bindingefficiencies of 52 and 53% are significantly less efficient than theadsorbents according to the invention which have average bindingefficiencies in the range from 75-95%.

4. 2-Mercaptobenzoic Acid as the Ligand

Isolation of Immunoglobulins Under Different Binding and WashingConditions.

As is indicated from the results in table I 2-mercaptobenzoic acid seemsto be a very interesting ligand for isolation and purification ofmonoclonal antibodies from dilute culture supernatants. Further studiesof this solid phase matrix employing the “artificial culturesupernatant” as described in example 2 was therefore performed with theaim of establishing the optimal binding and washing conditions so as toachieve the maximal binding capacity as well as yield and purity of theantibody in the eluate.

2-mercapto-benzoic acid agarose was based on epichlorohydrin activated6% agarose beads from Hispanagar and synthesised as described in example1A and 1E. The ligand concentration was measured by two differentmethods and found to be 65 μmol/g wet, but drained matrix as determinedby elementary analysis and 60 μmol/g as determined by acid-basetitration of the immobilised benzoic acid part of the ligand.

Generally the experiments were performed according to the followingprocedure:

-   1) A small aliquot of 2-mercaptobenzoic acid agarose was washed with    water (all water unless otherwise stated had the quality of Milli Q    water) on a sintered glass funnel by gentle suction followed by    draining of the interstitial water by light suction for one minute.-   2) 0.4 gram of wet, but drained matrix was then weighed into a test    tube followed by the addition of 10 ml “artificial culture    supernatant” having a specific pH-value for that particular    experiment. With or without any further additives the suspension was    hereafter incubated on a roller mixer for two hours at room    temperature to ensure efficient binding of the immunoglobulin.-   3) Following incubation the matrix was transferred to a column with    a 5 mm inner diameter, drained for excess “artificial culture    medium” and washed according to a scheme specific for the particular    experiment. Washing was performed by adding 4×4 ml washing buffer to    the column and collecting the run-through from the column in one    fraction.-   4) The final elution of bound immunoglobulin was performed with a    specific elution buffer by addition of 4×2.5 ml buffer to the column    and collecting the eluate in one fraction. No pumps were employed in    the experiments, all columns were run by gravity (at an approximate    flow rate of 0.5-1.0 ml/min).-   5) Analyses were performed to determine the relative distribution of    immunoglobulin between the non-bound fraction in the supernatant    after binding, the washing fraction(s) and the eluate. This was done    by single radial immunodiffusion (as described in D. Catty and C    Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using rabbit anti mouse immunoglobulins as the    precipitating antibody (DAKO A/S, Denmark, Cat. no.: Z 109).

The binding capacity was then calculated from the amount of non-boundimmunoglobulin present in the supernatant and expressed as a percentageof the total amount added to the matrix in the raw material.

The yield was calculated as the percentage of the added immunoglobulinfound in the eluate fraction (i.e. a yield of 100% is equal to thepresence of 1 mg IgG in the eluate).

The purity of the eluted immunoglobulin was analysed by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide electrophoresis) under reducingconditions followed by staining of the protein bands with coomassiebrilliant blue. (Precast gel 4-20% tris-glycine, 1 mm cat. no.: EC6025,running 1 hour at 30 mA; tris-glycine SDS running buffer cat no.:LC2675; tris-glycine sample buffer cat no.: LC2676; coomassie stainingkit LC6025 all chemicals from Novex, USA)

The degree of purity as expressed in percent of the total proteincontents was determined by scanning and image processing of thecoomassie stained and dried polyacrylamide gel. For this purpose weemployed the CREAM system available from Kem-En-Tec A/S, Denmark (cat.no.: 6010+6050).

4A) The Effect of Performing Binding at Different pH-Values

The following experiment was performed to establish the pH-range inwhich the 2-mercapto-benzoic acid matrix would bind immunoglobulinsefficiently from the “artificial culture supernatant”. As was shown inTable I example 2, this matrix binds 100% at pH 4.5 and 0% at pH 7.0. Inthis experiment the binding efficiency, yield and purity of the eluateis determined when binding is performed in the pH range 3.0-6.5. In allinstances the washing buffer used was 10 mM citric acid buffer adjustedto the same pH as the binding pH with 1 M sodium hydroxide. The elutionbuffer used was in all instances 0.05 M boric acid/NaOH+0.5 M sodiumchloride pH 8.6.

Results:

pH of binding percent binding Yield (%) Purity (%) 3.0 100 90 <5 3.5 10095 <5 4.0 100 100 <5 4.5 100 100 <5 5.0 95 95 5 5.5 40 40 10 6.0 0 0 6.50 0

As can be seen from the table efficient binding is achieved at pH-valuesbelow 6.0 reaching 100% at pH 4.5. At the same time there is anindication that a relatively higher purity may be obtained if thebinding step is performed at a higher pH than 4.5.

4B) The Effect of Different Washing Procedures/pH in Washing Buffer

A series of tests were performed with the aim of optimising the purityof the eluate while maintaining the yield at a high level. For thispurpose a range of different washing procedures were testes All testswere performed with pH 4.5 as the pH of binding and all eluates wereperformed with 0.05 M boric acid/NaOH+0.5 M NaCl pH 8.6.

Results:

Washing buffer: purity (%) yield (%) 10 mM citric acid/NaOH pH 4.5 <5100 10 mM citric acid/NaOH pH 5.5 5 95 10 mM citric acid/NaOH pH 6.5 1580 20 mM TRIS/HCl pH 7.5 20 70 20 mM TRIS/HCl pH 8.5 20 60

As can be seen from the table the purity of the eluate may be increasedby washing with a higher pH, but an increase in pH above pH 5.5decreases the yield significantly.

4C) The Effect of Different Washing Procedures/Lyotropic Salts at HighpH

Experiments were performed as described in 3B except that a series ofwashing buffers containing different lyotropic salts at pH 8.0 weretested for their ability to improve the purity of the eluate withoutsignificantly decreasing the yield.

Results:

Washing buffer: purity (%) yield (%) 0.7 M ammonium sulfate/NaOH pH 8.0ND <10 0.9 M ammonium sulfate/NaOH pH 8.0 25 30 1.0 M ammoniumsulfate/NaOH pH 8.0 25 80 1.1 M ammonium sulfate/NaOH pH 8.0 20 95 1.3 Mammonium sulfate/NaOH pH 8.0 20 95 0.8 M potassium phosphate pH 8.0 2095 1.0 M potassium phosphate pH 8.0 15 95 0.9 M sodium sulfate/NaOH + 2095 0.05 M sodium bicarbonate pH 8.0 1.0 M sodium sulfate/NaOH + 20 950.05 M sodium bicarbonate pH 8.0 1.0 M sodium chloride + ND 0 0.05 Mpotassium phosphate pH 8.0 2.0 M sodium chloride + ND 0 0.05 M potassiumphosphate pH 8.0 4.0 M sodium chloride + 20 80 0.05 M potassiumphosphate pH 8.0

The results indicate that the presence of lyotropic salts in the washingbuffer combined with a higher pH than the binding pH may increase thepurity of the eluate significantly without decreasing the yield. It isalso evident that a certain concentration of the lyotropic salt isnecessary to obtain this result. Too low concentrations results in lossof immunoglobulin in the washing fraction, resulting in very low yields.As can be seen the necessary concentration is dependent on the nature ofthe lyotropic salt, e.g. ammonium sulfate which is considered a stronglylyotropic salts according to the Hofmeister series (see Gagnon citedherein) need only to have a concentration of about 1.0-1.1 M, to ensurea high yield in the eluate, while sodium chloride, which is considered apoor weakly lyotropic salt according to the Hofmeister series, needs tohave a concentration of about 4 M before the yield is increasing to anacceptable level.

4D) The Effect of Different Washing Procedures/Different Additives

The effect of adding detergents and other additives to the washingbuffer was investigated in tests performed as described above (example4B and 4C).

Results:

Washing buffer: purity (%) yield (%) 0.01 M citric acid/NaOH pH 6.5 + 5080 3 mg/ml octyl sulfate 0.01 M citric acid/NaOH pH 5.8 + 70 90 0.05mg/ml bromophenol blue 1.0 M ammonium sulfate/NaOH pH 7.5 + 80 80 10mg/ml octane sulfonic acid 1.0 M ammonium sulfate/NaOH pH 8.0 + 60 80 5mg/ml sodium laurylsarcosinate 1.0 M ammonium sulfate/NaOH pH 8.0 + 8070 5 mg/ml octane sulfonic acid + 5 mg/ml sodium laurylsarcocinate 0.9 Mpotassium phosphate pH 9.2 + 80 80 5 mg/ml octane sulfonic acid 0.9 Mpotassium phosphate pH 9.2 + 60 90 5 mg/ml hexane sulfonic acid 1.0 Mammonium sulfate/NaOH pH 8.0 + 25 90 5 mg/ml tween 20 1.0 M ammoniumsulfate/NaOH pH 8.0 + 25 80 5 mg/ml pluronic F68

The results from these experiments clearly indicate the positive effecton the purity of the eluate obtained by washing the matrix with bufferscontaining a negatively charged detergent (e.g. octane sulfonic acid,hexane sulfonic acid, octyl sulfate and sodium laurylsarcosinate), whilethe addition of uncharged detergents such as Tween 20 and pluronic F-68seems to have little or no effect on the purity of the elutedimmunoglobulin. Likewise it is shown that bromophenol blue, which isknown to have a high affinity for binding to albumin (an unwantedimpurity) also has a significant effect on the purity withoutcompromising the yield of product. Furthermore the obtained effect seemsto be independent of whether the washing buffer comprises highconcentrations of lyotropic salts or not as well as the choice oflyotropic salt used, if present.

4E) The Effect of Different Additives During Binding

The following experiments were performed to investigate the effect onpurity and yield of the addition of different detergents and otherchemical substances to the “artificial culture supernatant” during theincubation with the 2-mercapto-benzoic acid agarose. For all tests thepH of binding was pH 5.0, the washing buffer used was 1.1 M ammoniumsulfate/NaOH pH 8.0 and the elution buffer was 0.05 M boricacid/NaOH+0.5 M sodium chloride pH 8.6. The experiments were otherwiseperformed as described in the general procedure above.

Results:

Substance added: purity (%) yield (%) None 25 95 5 mg/ml Tween 20 30 8010 mg/ml benzoic acid 25 50 5 mg/ml 1-octyl-2-pyrrolidone 20 80 5 mg/mlN-octanoyl-N-methylglucamine 20 80 1 mg/ml lauryl sulfobetaine 20 80 5mg/ml lauryl sulfobetaine ND 0 5 mg/ml suberic acid 25 80 5 mg/mlsebacic acid 25 80 5 mg/ml octane sulfonic acid 25 90 5 mg/ml caproicacid 60 90 5 mg/ml caprylic acid 70 80 0.5 mg/ml sodiumlaurylsarcosinate 70 90 1.0 mg/ml sodium laurylsarcosinate 85 90 2.0mg/ml sodium laurylsarcosinate 90 70 1 mg/ml bromophenol blue 80 90

The results indicate that the addition of certain negatively chargeddetergents (or amphophilic substances) to the “artificial culturesupernatant” prior to the incubation with 2-mercapto-benzoic acidagarose has a significant influence on the final purity of the eluate.This is for example the case for substances such as caproic and caprylicacid as well as lauryl sarcosinate, while other negatively chargedsubstances such as benzoic acid, lauryl sulfobetaine, suberic acid,sebacic acid and octane sulfonic acid seems to have very little effectin the concentrations tested. It is also noted that the neutraldetergents Tween 20 and the positive detergent 1-octyl-N-methylglucamineseems to have no effect either.

4F) The Effect of Different Washing Buffers in Combination with theAddition of Sodium Laurylsarcosinate to the Raw Material

The following example demonstrates the effect of combining the additionof a negatively charged detergent to the raw material with a series ofdifferent washing buffer compositions. In all experiments there is added1 mg/ml sodium lauryl sarcosinate to the “artificial culturesupernatant” prior to mixing with the 2-mercapto-benzoic acid agarose,pH of binding were pH 5.0 and the elution buffer were in all cases 0.05M boric acid/NaOH+0.5 M sodium chloride pH 8.6. Otherwise the generalprocedure described above was followed.

Results:

Washing buffer: purity (%) yield (%) water 45 90 0.001 M sodium citratepH 6.0 45 90 0.001 M sodium citrate pH 6.5 50 90 0.001 M sodium citratepH 7.0 50 90 0.001 M potassium phosphate pH 7.5 50 90 0.001 M sodiumcitrate pH 6.5 + 50 80 5% monopropylene glycol 0.001 M sodium citrate pH6.5 + 45 95 20% monopropylene glycol 1.0 M ammonium sulfate/NaOH pH 7.560 85 1.0 M ammonium sulfate/NaOH pH 7.0 60 90 0.9 M ammoniumsulfate/NaOH pH 7.0 60 755. 4-Amino-Benzoic Acid as the LigandIsolation of Monoclonal Antibodies Under Different Conditions

4-amino-benzoic acid is another aromatic acid according to the inventionthat seems to be very interesting for use in monoclonal antibodypurification (table I, example 2). The following tests demonstrates theinfluence of different binding and washing conditions on the performanceof 4-amino-benzoic acid agarose based on 6% agarose from Hispanagar andsynthesised according to example 1A and 1E. The matrix used was analysedby elemental analysis and determined to have a content of 69 μmol4-amino-benzoic acid groups per ml wet, but drained matrix.

The tests were performed as described in the general procedure inexample 4.

5A) The Effect of Performing Binding at Different pH-Values

The following experiment was performed to establish the pH-range inwhich the 4-amino-benzoic acid matrix would bind immunoglobulinsefficiently from the “artificial culture supernatant”. As was shown inTable I example 2, this matrix binds 90% at pH 4.5 and 0% at pH 7.0. Inthis experiment the binding efficiency, yield and purity of the eluateis determined when binding is performed in the pH-range 4.0-6.5. In allinstances the washing buffer used was 10 mM citric acid buffer adjustedto the same pH as the binding pH with 1 M sodium hydroxide. The elutionbuffer used was in all instances 0.05 M boric acid/NaOH+0.5 M sodiumchloride pH 8.6.

Results:

pH of binding percent binding Yield (%) Purity (%) 4.0 100 90 10 4.5 9095 10 5.0 60 55 20 5.5 20 ND ND 6.0 0 ND ND 6.5 0 ND ND

As can be seen from the table efficient binding is achieved at pH-valuesbelow 5.5 reaching 90% at pH 4.5. At the same time there is anindication that a relatively higher purity may be obtained if thebinding step is performed at a higher pH than 4.5.

5B) The Effect of Different Washing Procedures/pH in Washing Buffer

A series of tests were performed with the aim of optimising the purityof the eluate while maintaining the yield at a high level. For thispurpose a range of different washing procedures were tested. All testswere performed with pH 4.5 as the pH of binding and all eluates wereperformed with 0.05 M boric acid/NaOH+0.5 M NaCl pH 8.6.

Results:

Washing buffer: purity (%) yield (%) 10 mM citric acid/NaOH pH 4.5 10 9010 mM citric acid/NaOH pH 5.5 25 90 10 mM citric acid/NaOH pH 6.0 60 8010 mM citric acid/NaOH pH 6.5 75 55

As can be seen from the table the purity of the eluate may be increasedby washing with a higher pH, but an increase in pH above pH 6.0decreases the yield significantly.

6. 2-Mercapto-Nicotinic Acid

Isolation of Monoclonal Antibodies Under Different Conditions

2-mercapto-nicotinic acid is another aromatic acid according to theinvention that seems to be very interesting for use in monoclonalantibody purification (table I, example 2). The following testsdemonstrates the influence of different binding and washing conditionson the performance of 2-mercapto-nicotinic acid agarose based onepichlorohydrin activated 6% agarose from Hispanagar and synthesisedaccording to example 1A and 1E. The matrix used was analysed byelemental analysis and determined to have a content of 63 μmol2-mercapto-nicotinic acid groups per ml wet, but drained matrix.

The tests were performed as described in the general procedure inexample 4.

In these two tests the effect of varying binding pH on yield and purityof the resulting eluate was investigated while keeping washing andelution conditions constant. In both instances the washing buffer was1.1 M ammonium sulfate/NaOH pH 8.0+5 mg/ml octyl sulfate and the elutionbuffer was 0.05 M boric acid/NaOH pH 8.6+0.5 M sodium chloride.

The “artificial culture supernatant” was adjusted to pH 4.5 and 5.0 with1 M hydrochloric acid respectively and no further additions were made.

Results:

Binding pH Yield, % Purity, % 4.6 85 80 5.0 75 95

As can be seen this matrix provides an excellent yield of immunoglobulinin the eluate at both binding pH-values while the purity of the elutedimmunoglobulin is significantly increased by raising the binding pH frompH 4.5 to pH 5.0.

Effect of Adding Sodium Lauryl Sarcosinate to the Raw Material

In the following tests the effect of adding different amounts of sodiumlauryl sarcosinate to the “artificial culture supernatant” at twodifferent binding pH-values is investigated. In all tests the washingbuffer used was 1.1 M ammonium sulfate/NaOH pH 7.5 and the elutionbuffer was 0.05 M boric acid/NaOH pH 8.6+0.5 M sodium chloride.

Prior to mixing with the solid phase matrix the “artificial culturesupernatant” was added sodium lauryl sarcosinate to three differentconcentrations and then adjusted to pH 4.5 and 5.0 with 1 M hydrochloricacid respectively.

Results

Concentration of SLS, mg/ml Yield, % Purity, % Binding at pH 4.5 0.5 9050 1.0 90 65 1.5 65 85 Binding at pH 5.0: 0.5 90 50 1.0 90 85 1.5 40 >95SLS = Sodium Lauryl Sarcosinate7. 2-Mercapto-benzimidazoleIsolation of Monoclonal Antibodies

As is indicated in Table I, 2-mercaptobenzimidazole represents anothervery interesting group of ligands (the benzimidazoles, benzoxazoles andbenzothiazoles) for immunoglobulin isolation. The following exampleillustrates the application of this ligand for binding and isolation ofimmunoglobulins from the “artificial culture supernatant” described inexample 2.

2-mercapto-benzimidazole agarose was based on epichlorohydrin activated6% agarose beads from Hispanagar and synthesised as described in example1A and 1E. The ligand concentration was measured by elemental analysisand found to be 69 μmol/g wet, but drained matrix.

In the following tests the yield and purity obtained by incubation ofthe 2-mercaptobenzimidazole agarose with “artificial culturesupernatant” containing different concentrations of added polyvinylpyrrolidone is determined by following the general procedure describedin example 4. The pH of binding was adjusted to pH 7.5 with hydrochloricacid, the washing buffer was 0.01 M potassium phosphate+0.5 M sodiumchloride pH 7.5 and the elution buffer was 0.01 M citric acid/NaOH pH3.5.

Results

Concentration of PVP, mg/ml Yield, % Purity, % 0.0 95 25 0.5 80 70 1.070 80 2.0 40 90 4.0 5 ND PVP: polyvinyl pyrrolidone

The results indicate that 2-mercapto-benzimidazole agarose is able tobind almost all of the applied monoclonal antibody (i.e. giving a yieldof 95%) and at the same time give an eluate which is substantiallypurified. The purity can even be increased by adding substances such aspolyvinyl pyrrolidone.

8. Stability at High pH

2-mercapto-benzimidazole was coupled to epichlorohydrin activated 6%agarose beads (Hispanagar) prepared as described in example 1A as wellas to divinyl sulfone activated 6% agarose beads (Hispanagar) preparedaccording to example 1D. Both coupling procedures were according toexample 1E.

The contents of 2 mercapto-benzimidazole of the two matrices weredetermined by elemental analysis and found to be 69 μmol/g wet (drained)matrix and 42 μmol/ml wet (drained) matrix respectively.

Both matrices were tested for their stability towards incubation with 1M sodium hydroxide by following the procedure described below:

Standard Stability Test

-   I) Approximately 1000 mg of the matrix to be tested is washed with    100 ml demineralised water on a sintered glass funnel followed by    suction draining for 60 seconds. 500 mg of wet (drained) solid phase    matrix is weighed into a 10.0 ml test tube labelled “NaOH” and 9.0    ml 1 M sodium hydroxide is added followed by mixing gently for 1    min. Another 500 mg of wet (drained) solid phase matrix is weighed    into a 10 ml test tube labelled “Water” and 9.0 ml water is added    followed by gentle mixing for 1 min.

The test tubes are closed tightly with stoppers stored dark at roomtemperature (20-25° C.) for 7 days.

The matrices are then washed separately with 200 ml water on a sinteredglass funnel followed by suction draining for 60 seconds.

-   II) Each of the solid phases matrices are tested in the “Standard    Immunglobulin Binding Test” defined in Example 2.

The stability of the solid phase matrix towards 1 M sodium hydroxide isthen calculated and expressed as a percentage compared to the controlwhich has only been incubated in water according to the followingformula:Stability=(percentage bound of NaOH treated matrix/percentage bound ofcontrol)×100%Results

Solid phase matrix Stability, %2-mercapto-benzimidazole-epichlorohydrin-agarose 982-mercapto-benzimidazole-divinyl sulfone-agarose 0

The results indicate that matrices produced with divinyl sulfoneactivated agarose have poor stability in 1M NaOH, whereas epoxyactivated agarose gives stable solid phase matrices. It is furthermoredemonstrated that 2-mercapto-benzimidazole is a stable ligand in itself.

9. Isolation of Polyclonal Antibodies from Different Species

2-mercaptobenzioc Acid as the Ligand

The following example illustrates the binding efficiency of2-meraaptobenzoic acid agarose towards polyclonal antibodies fromdifferent species, as well as yield and purity of the antibody in theeluate. For the study sera from 5 different species were used.

Polyclonal antibodies: The polyclonal antibodies used originated fromnormal sera from the following species: goat, horse, rabbit, swine andhuman. The sera were obtained from freshly drawn blood by mildcentrifugation after coagulation for 24 hours at room temperature.

Solid phase matrix 2-mercaptobenzoic acid agarose was based on epoxyactivated 6% agarose beads from Hispanagar and synthesised as describedin example 1A and 1E. The ligand concentration was measured by elementalanalysis and found to be 65 μmol/gram wet, but drained matrix.

The solid phase matrix was tested for its polyclonal antibody bindingefficiency in a column according to the following procedure:

-   1) The matrix was washed with water on a sintered glass funnel and    finally drained 1 gram of wet, but drained solid phase matrix was    weighed into a small column (inner diameter of 5 mm). The matrix was    washed with 5 ml of buffer. (10 mM sodium citrate pH 5.0.). 1 ml of    the sample (adjusted to pH 5.0 with 1 M hydrochloric acid) was    applied to the column. The column was washed with 20 ml of washing    buffer I (1.1 M ammonium sulfate pH 8.0 containing 5 mg/ml sodium    1-octanesulfonate). The column was washed with 5 ml of washing    buffer II (1.1 M ammonium sulfate pH 8.0). The matrix was eluted    with 10 ml of elution buffer (0.05 M boric acid/NaOH+0.5 M sodium    chloride pH 8.6). No pumps were employed in the experiments, all    columns were run by gravity (at an approximate flow rate of 0.5-1.0    ml/min).-   2) Analyses were performed to determine the relative distribution of    immunoglobulin between the non-bound fraction in the run-through    after binding, the washing fraction(s) and the eluate. This was done    by single radial immunodiffusion (as described in D. Catty and C    Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using species specific anti-immunoglobulins as the    precipitating antibodies.

The binding capacity was then calculated from the amount of non-boundimmunoglobulin present in the run-through and expressed as a percentageof the total amount added to the matrix in the raw material.

The yield was calculated as the percentage of the added immunoglobulinfound in the eluate fraction.

The purity of the eluted immunoglobulin was analysed by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide electrophoresis) under reducingconditions followed by staining of the protein bands with coomassiebrilliant blue. (Precast gel 4-20% tris-glycine, 1 mm cat. no.: EC6025,running 1 hour at 30 mA; tris-glycine SDS running buffer cat. no.:LC2675; tris-glycine sample buffer cat. no.: LC2676; coomassie stainingkit LC6025 all chemicals from Novex, USA)

The degree of purity as expressed in percent of the total proteincontents was determined by scanning and image processing of thecoomassie stained and dried polyacrylamide gel. For this purpose weemployed the CREAM system available from Kem-En-Tec A/S, Denmark (cat.no.: 6010+6050).

Results

binding capacity Serum (%) Yield (%) Purity (%) Goat 60 50 60 Swine 6060 70 Rabbit 80 60 90 Horse 60 40 80 Human 70 60 7510. Isolation of IgG from Bovine Serum2-mercaptobenzimidazol as the Ligand

The following example illustrates that it is possible to isolate andpurify IgG from bovine serum with 2-mercaptobenzimidazol as the ligand.

Bovine serum: The bovine serum used was normal serum. The serum wasobtained from freshly drawn blood by mild centrifugation aftercoagulation for 24 hours at room temperature.

Solid phase mad 2-mercaptobenzimidazol agarose was based on epoxyactivated 6% agarose beads from Hispanagar and synthesised as describedin example 1A and 1E. The ligand concentration was measured by elementalanalysis and found to be 69 μmol/g wet, but drained matrix.

The solid phase matrix was tested for it's polyclonal antibody bindingefficiency in a column according to the following procedure:

-   1) The matrix was washed with water on a sintered glass funnel and    finally drained. 2 gram of wet, but drained solid phase matrix is    weighed into a small column (inner diameter of 5 mm). The matrix was    washed with 5 ml of buffer. (10 mM sodium citrate pH 7.0.). 2 ml of    bovine serum was applied to the column. The column was washed with    10 ml of washing buffer (10 mM sodium citrate, 0.25 M NaCl pH 7.0).    The matrix was eluted with 20 ml of elution buffer (10 mM sodium    citrate pH 3.0). The flow rate was 1.0 ml/min.-   2) Analyses were performed to determine the relative distribution of    immunoglobulin between the non-bound fraction in the run-through    after binding, the washing fraction(s) and the eluate. This was done    by single radial immunodiffusion (as described in D. Catty and C    Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using rabbit anti cow immunoglobulins (DAKO, Denmark    cat. no.: Z247) as the precipitating antibody.

The binding capacity was then calculated from the amount of non-boundimmunoglobulin present in the run-through and expressed as a percentageof the total amount added to the matrix in the raw material.

The yield and purity was determined as described in example 4.

Results

Binding capacity 85% Purity 80% Yield 85%11. Isolation of Immunoglobulins from Egg Yolk2-mercaptobenzimidazol as the Ligand

The following example illustrates that it is possible to isolateimmunoglobulins from egg yolk with 2-mercaptobenzimidazol as the ligand.

Egg yolk Egg yolks (from normal chicken eggs) were diluted 1:1 with 0.25M NaCl. The sample was centrifuged in 20 minutes at 10.000 rpm.

Solid phase matrix: 2-mercaptobenzimidazol agarose was based on epoxyactivated 6% agarose beads from Hispanagar and synthesised as describedin example 1A and 1E. The ligand concentration was measured by elementalanalysis and found to be 69 μmol/gram wet, but drained matrix.

The solid phase matrix was tested for it's efficiency to bindimmunoglobulins from egg yolk in a column according to the followingprocedure:

-   1) The matrix was washed with water on a sintered glass funnel and    finally drained 2 gram of wet, but drained solid phase matrix is    weighed into a small column (inner diameter of 5 mm). The matrix was    washed with 10 ml of buffer. (10 mM KH₂PO₄ 6.1.). 4 ml of the sample    was applied to the column. The column was washed with 10 ml washing    buffer (10 mM KH₂PO₄ 6.1). The matrix was eluted with 16 ml of    elution buffer (10 mM sodium citrate pH 3.5).-   2) Analyses were performed to determine the relative distribution of    immunoglobulin between the non-bound fraction in the run-through    after binding, the washing fraction(s) and the eluate. This was done    by single radial immunodiffusion (as described in D. Catty and C    Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using rabbit anti chicken IgG (Sigma, USA cat. no.:    C-6409) as the precipitating antibody.

The binding capacity was then calculated from the amount of non-boundimmunoglobulin present in the run-through and expressed as a percentageof the total amount added to the matrix in the raw material.

The yield and purity was determined as described in example 4.

Results

Binding capacity 80% Purity 60% Yield 80%12. Depletion of IgG and Haemoglobin from Fetal Calf Serum

The following example illustrates the efficiency of some of the solidphases according to the invention to deplete IgG and haemoglobin fromfetal calf serum. The study was designed to determine the bindingefficiency during batch incubation with 14 different solid phases.

Fetal calf serum: The fetal calf serum was obtained from freshly drawnblood by mild centrifugation after coagulation for 24 hours at roomtemperature.

Solid phase The following solid phases were used 2-mercaptobenzimidazolagarose, thiophenol agarose, 4-chlorothiophenol agarose,2-aminothiophenol agarose, 4-methylmercaptoaniline agarose,2-mercapto-5-nitrobenzimidazole agarose, benzylmercaptan agarose,2-chlorophenol agarose, 3-chlorophenol agarose, 4chlorophenol agarosc,2-mercaptobenzoxazol agarose, 2-mercaptopyridine agarose,2,5-dimercapto-1,3,4,-thiadiazol agarose,6-ethoxy-2-mercaptobenzothiazol agarose. All these agaroses were basedon epoxy activated 6% agarose beads from Hispanagar and synthesised asdescribed in example 1A and 1E. The ligand concentration was measuredfor all matrices by elemental analysis and found to be in the range of60-70 μmol/g wet, but drained matrix.

The solid phase matrices were tested for their efficiency to deplete IgGand haemoglobin from fetal calf serum according to the followingprocedure:

-   1) The matrix was washed with 0.25 M NaCl on a sintered glass funnel    and finally drained 0.5 gram of wet, but drained solid phase matrix    is weighed into a test tube and added 5 ml of fetal calf serum. The    suspension was then incubated on a roller mixer for two hours at    room temperature.-   2) After incubation the test tube was centrifuged for 5 min. at 2000    RPM to sediment the matrix and a sample of the supernatant was taken    out for determination of the amount of IgG left in the serum. This    was done by single radial immunodiffusion (as described in D. Catty    and C Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using rabbit anti cow immunoglobulins (DAKO, Denmark,    cat. no.: Z247) as the precipitating antibody.-   3) The concentration of haemoglobin was measured    spectrophotometrical at 414 nm. The percentage of haemoglobin left    unbound in the serum is calculated as:    (Abs_(414 nm, absorbed fetal calf serum)/Abs_(414 nm, fetal calf serum)×)100%    Results

% haemoglobin % IgG left Sample left in serum in serum2-mercaptobenzimidazol agarose 60 30 Thiophenol agarose 60 504-chlorothiophenol agarose 40 40 2-aminothiophenol agarose 60 304-methylmercaptoaniline agarose 90 60 2-mercapto-5-nitrobenzimidazoleagarose 30 30 Benzylmercaptan agarose 70 60 2-chlorophenol agarose 80 603-chlorophenol agarose 70 50 4-chlorophenol agarose 75 502-mercaptobenzoxazol agarose 75 50 2-mercaptopyridine agarose 75 602,5-dimercapto-1,3,4,-thiadiazol agarose 10 406-ethoxy-2-mercaptobenzothiazol agarose 60 6013. Isolation of Trypsinogen and Chymotrypsinogen from Bovine Pancreaswith 2-Mercapto-Menzoic Acid Agarose

The following example illustrates the use of 2-mercapto-benzoic acidagarose as a suitable matrix for isolation and purification ofproteases, e.g. trypsin and chymotrypsin from bovine pancreas.

Pancreas extract: The two proteases were isolated as the proenzymestrypsinogen and chymotryp-sinogen from a bovine pancreas extractproduced by extraction with sulfuric acid as described in M. Laskowski,Methods in Enzymology, vol. II, pp 9-10, 1955. After extraction thesuspension was adjusted to pH 2.5 by addition of 2 M sodium hydroxideand clarified by filtration and centrifugation for 30 min. at 4000 RPM.Just prior to purification the extract was adjusted to pH 4.5 with 2 MNaOH and centrifugated at 4000 rpm for 5 minutes the supernatant wascollected.

Solid phase matrix: 2-mercapto-benzoic acid agarose was based onepichlorohydrin activated 6% agarose beads from Hispanagar andsynthesised as described in example 1A and 1E. The ligand concentrationwas measured by two different methods and found to be 65 μmol/g wet, butdrained matrix as determined by elementary analysis and 60 μmol/g asdetermined by acid-base titration of the immobilised benzoic acid partof the ligand.

The solid phase matrix was tested for the efficiency to bind trypsin andchymotrypsin according to the following procedure:

-   1) The matrix was washed with water on a sintered glass funnel and    finally drained 2,5 gram of wet, but drained solid phase matrix is    weighed into a column (inner diameter of 5 mm). The matrix was    washed with 10 ml buffer (10 mM sodium citrate pH 4.5) 50 ml of the    extract was applied to the column. The matrix was washed with 15 ml    of washing buffer (10 mM sodium citrate pH 4.5). The matrix was    eluted with 10 ml elution buffer (50 mM boric acid, 0.5 M NaCl pH    8.7).-   2) The purity of the eluate was analysed by SDS-PAGE (sodium dodecyl    sulfate polyacrylamide electrophoresis) under reducing conditions    followed by staining of the protein bands with coomassie brilliant    blue. (Precast gel 4-20% tris-glycine, 1 mm cat. no.: EC6025,    running 1 hour at 30 mA, tris-glycine SDS running buffer cat. no.:    LC2675; tris-glycine sample buffer cat. no.: LC2676; coomassie    staining kit LC6025 all chemicals from Novex, USA).    Results

Total amount of protein in eluate 65 mg Trypsin in eluate 35%Chymotrypsin in eluate 20%

As can be seen from these results it is surprisingly found that thistype of ligand i.e. aromatic ligands comprising an acidic groupaccording to the invention, here represented by 2mercapto-benzoic acidas the specific ligand, are able to bind very efficiently proteins suchas proteases at relatively low pH values and at relatively high ionicstrength (i.e. approx 0.25 in ionic strength).

14. Purification of Immunoglobulins from Horse Serum

The following example illustrates the use of 2-mercapto-benzimidazolecoupled to agarose beads for purification of immunoglobulins from horseserum. It further illustrates the use of different elution conditionswith this type of matrix.

Horse serum: The horse serum was obtained from freshly drawn blood bymild centrifugation after coagulation for 24 hours at room temperature.

Solid phase matrix: 2-mercapto-benzimidazole agarose was produced asdescribed in example 1A and 1E. The ligand concentration was determinedto be 69 μmol/g wet but suction drained matrix.

Procedure:

-   1) The matrix was washed with water on a sintered glass funnel and    finally drained. 2 g of wet, but drained 2-mercapto-benzimidazole    agarose is weighed into a small column (inner diameter of 5 mm). The    matrix was washed with 5 ml of 10 mM potassium phosphate, pH 7.0. 2    ml of horse serum adjusted to pH 7.0 with 0.1 M HCl was applied to    the column. The column was washed with 10 ml of washing buffer (10    mM potassium phosphate, 0.1 M NaCl, pH 7.0). The matrix was eluted    with 20 ml of elution buffer (see below). The flow rate was 1.0    ml/min.

This procedure was followed in three identical experiments except forthe use of three different elution buffers:

-   Elution buffer A=20 mM sodium citrate pH 3.0-   Elution buffer B=50 mM ethanol amine/HCl pH 11.0-   Elution buffer C=10 mM potassium phosphate pH 7.0+30% v/v    1,2-propane diol-   2) Analyses were performed to determine the relative distribution of    immunoglobulin between the non-bound fraction in the run-through    after binding, the washing fraction(s) and the eluate. This was done    by single radial immunodiffusion (as described in D. Catty ad C    Raykundalia “Antibodies—a practical approach” Vol I, pp.    137-168, 1988) using rabbit anti horse immunoglobulin G (Sigma, USA,    cat. no.: H-9015) as the precipitating antibody.

The binding capacity was then calculated from the amount of non-boundimmunoglobulin present in the run-through and expressed as a percentageof the total amount added to the matrix in the raw material. The yieldand purity was determined as described in example 4.

Results:

Elution Elution Elution buffer A buffer B buffer C Binding capacity % 8585 85 Yield % 80 85 80 Purity % 90 90 85

The results indicated that 2-mercapto-benzimidazole agarose is anefficient solid phase matrix for purification of horse immunoglobulinsand that elution may be performed with either weakly acidic or weaklybasic buffers or alternatively with a neutral buffer comprising anon-toxic organic solvent such as 1,2-propanediol without compromisingyield and purity of the eluted immunoglobulin.

15. Bovine Serum Albumin Binding Efficiency by Different Solid PhaseMatrices

The following example illustrates the efficiency of different solidphase matrices in a standard binding assay for bovine serum albumin.

Solid phases: A selected range of solid phase matrices were produced onthe basis of epichlorohydrin activated agarose beads from Hisapanagar asdescribed in example 1A and 1E. The ligands tested are listed in thetable below.

Bovine serum albumin solution pH 4.0 (BSA pH 4.0): Purified bovine serumalbumin (Biofac A/S, Denmark) was dissolved to a final concentration of10 mg/ml in 20 mM sodium citrate pH 4.0+0.2 M sodium chloride.

Bovine serum albumin solution pH 7.0 (BSA pH 7.0): Purified bovine serumalbumin (Biofac A/S, Denmark) was dissolved to a final concentration of10 mg/ml in 20 mM sodium citrate pH 7.0+0.2 M sodium chloride.

Procedure:

Standard Albumin Binding Assay:

The solid phase matrices were washed with 10 volumes of demineralisedwater on a vacuum suction filter and drained by gentle suction for 1min. Two samples of 1.0 g suction drained matrix were then weighed intotwo 10 ml test tubes followed by the addition of 6.0 ml of BSA pH 4.0 tothe first test tube and 6.0 ml BSA pH 7.0 to the second test tube. Two1.0 g samples of non-derivatised suction drained plain agarose beadsfrom Hispanagar were also added 6.0 ml of the two BSA solutions asnegative controls. The test tubes were then close with a stopper and thesuspension incubated on a roller mixer for 2 hours at room temperature(20-25° C.). The test tube was then centrifuged for 5 min. at 2000 RPMin order to sediment the matrix. The supernatants were then isolatedfrom the solid phase matrix by pipetting into a separate test tubes,avoiding the carry-over of any matrix particles and filtered through asmall non-adsorbing 0.2 μm filter (Millipore, USA). Following this adetermination of the concentration of non-bound BSA in the supernatantis performed by measuring the optical density (OD) at 280 nm on aspectrophotometer.

The amount of BSA bound to the matrices were then calculated accordingto the following formula:mg BSA bound per g suction drained matrix=(1−(OD of test supernatant/ODof control))×60

The precision of this method is better than +/−5%.

Results:

The table gives the amount of BSA bound in mg per gram wet, but suctiondrained matrix as a function of the coupled ligand and the pH of the BSAsolution.

Ligand coupled to solid phase matrix BSA pH 4.0 BSA pH 7.03-hydroxy-benzoic acid 22 0 4-hydroxy-benzoic acid 5 03,5-dihydroxy-benzoic acid 36 58 2,4-dihydroxy-benzoic acid 41 02-hydroxy-1-naphthalic acid 58 0 3-amino-benzoic acid 56 02-amino-benzoic acid 51 0 4-amino-benzoic acid 59 0 3,4-di-amino-benzoicacid 9 0 5-amino-iso-phthalic acid 17 51 1-amino-2-naphthol-4-sulfonicacid 21 52 p-coumaric acid 26 0 2-mercapto-benzoic acid 59 02-mercapto-nicotinic acid 30 0 5-mercapto-1-tetrazol-acetic acid 26 03-amino-1,2,4-triazol-5-carboxylic acid 6 302,5-di-mercapto-1,3,4-thiadiazol 0 20 2-amino-6-nitro-benzothiazol 28 462-mercaptobenzthiazol 42 45 Sulfa-thiazol 28 0 Sulfa-methizol 20 02-amino-pyridin 0 0 2-mercapto-pyridin 8 26 2-hydroxy-pyridin 0 192-mercapto-5-nitro-benzimidazol 58 47

1. A method for the isolation of proteins from a solution containing oneor more proteins, comprising the following operations: a) contacting asolution containing one or more proteins having a pH in the range of 2.0to 10.0 and a total salt content corresponding to an ionic strength ofat the most 2.0 with a solid phase matrix comprising a functionalizedmatrix backbone carrying a plurality of functional groups of thefollowing formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton: andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein the pH of the eluent is in the range of 6.0 to
 11. 2. A methodfor the isolation of proteins from a solution containing one or moreproteins, comprising the following operations: a) contacting a solutioncontaining one or more proteins having a pH in the range of 2.0 to 10.0and a total salt content corresponding to an ionic strength of at themost 2.0 with a solid phase matrix comprising a functionalized matrixbackbone carrying a plurality of functional groups of the followingformulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton; andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein the washing of the solid phase matrix (operation (c)) compriseswashing with an aqueous solution comprising a negatively chargeddetergent.
 3. A method according to claim 2, wherein the washing of thesolid phase matrix (operation (c)) comprises washing with an inorganicor organic salt buffer comprising a negatively charged detergent.
 4. Amethod for the isolation of proteins from a solution containing one ormore proteins, comprising the following operations: a) contacting asolution containing one or more proteins having a pH in the range of 2.0to 10.0 and a total salt content corresponding to an ionic strength ofat the most 2.0 with a solid phase matrix comprising a functionalizedmatrix backbone carrying a plurality of functional groups of thefollowing formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton: andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein the solution containing the proteins comprises a negativelycharged detergent.
 5. A method for the isolation of proteins from asolution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 10.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton; andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein the molecular weight of the ligand-spacer arm (SP1-L) is below250 Dalton.
 6. A method for the isolation of proteins from a solutioncontaining one or more proteins, comprising the following operations: a)contacting a solution containing one or more proteins having a pH in therange of 2.0 to 10.0 and a total salt content corresponding to an ionicstrength of at the most 2.0 with a solid phase matrix comprising afunctionalized matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton: andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix: whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein L is an aromatic radical selected from the group consisting of aphenyl, 1,2-phenylene, 1,3phenylene, 1,4-phenylene, 1,2,3-benzenetriyl,1,2,4-benzcnetriyl, 1,3,5-benzenetriyl, 1,2,3,4-benzenetetrayl,1,2,3,5-benzenetetrayl, 1,2,4,6-benzenetetrayl, and1,2,3,4,5-benzenepentayl.
 7. A method for the isolation of proteins froma solution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 10.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety: wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton; andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein L is an aminobenzoic acid selected from the group consisting of2-amino-benzoic acid; 2-mercapto-benzoic acid; 3-aminobenzoic acid;4-aminobenzoic acid; 4-amino-2-chlorobenzoic acid;2-amino-5-chlorobenzoic acid; 2-amino-4-chlorobenzoic acid;4-aminosalicylic acids; 5-aminosalicylic acids; 3,4-diaminobenzoicacids; 3,5:-diaminobenzoic acid; 5-aminoisophthalic acid; and4-aminophthalic acid.
 8. A method for the isolation of proteins from asolution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 10.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton; andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein L is a heteroaromatic compound comprising a substituent selectedfrom the group consisting of 2-amino-nicotinic acid,2-mercapto-nicotinic acid, 6-amino-nicotinic acid and2-amino-hydroxypyrimidine-carboxylic acid.
 9. A method for the isolationof proteins from a solution containing one or more proteins, comprisingthe following operations: a) contacting a solution containing one ormore proteins having a pH in the range of 2.0 to 10.0 and a total saltcontent corresponding to an ionic strength of at the most 2.0 with asolid phase matrix comprising a functionalized matrix backbone carryinga plurality of functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group selected from the groupconsisting of a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂), with the proviso that the molecularweight of the ligand-spacer arm (SP1-L) is at the most 500 Dalton; andthe ligand concentration is in the range of 10-120 μmol/ml hydrated,sedimented solid phase matrix; whereby at least a part of the proteinsbecomes bound to the solid phase matrix; then b) separating the solidphase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 10.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25% compared to a corresponding non-incubated matrix,wherein the spacer SP1is a short chain aliphatic biradical selected fromthe group of consisting of —CH₂—CH(OH)—CH₂-, -(CH₂)₃—O—CH₂—CH(OH)—CH₂-,or —CH₂—CH(OH)—CH₂—O—(CH₂) ₄—O—CH₂—CH(OH)—CH₂-.
 10. A method accordingto claims 1, 2, 3, 4, 5 or 9, wherein L is an aromatic radical selectedfrom benzene radicals and naphthalene radicals.
 11. A method accordingto claims 1, 2, 3, 4, 6, 7, 8 or 9, wherein the molecular weight of theligand-spacer arm (SP1-L) is below 250 Dalton.
 12. A method for theisolation of proteins from a solution containing one or more proteins,comprising the following operations: a) contacting a solution containingone or more proteins having a pH in the range of 2.0 to 10.0 and a totalsalt content corresponding to an ionic strength of at the most 2.0 witha solid phase matrix comprising a functionalized matrix backbonecarrying a plurality of functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinthe pH of the eluent is in the range of 6.0 to
 11. 13. A method for theisolation of proteins from a solution containing one or more proteins,comprising the following operations: a) contacting a solution containingone or more proteins having a pH in the range of 2.0 to 10.0 and a totalsalt content corresponding to an ionic strength of at the most 2.0 witha solid phase matrix comprising a functionalized matrix backbonecarrying a plurality of functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinthe washing of the solid phase matrix (operation (c)) comprises washingwith an aqueous solution comprising a negatively charged detergent. 14.A method according to claim 13, wherein the washing of the solid phasematrix (operation (c)) comprises washing with an inorganic or organicsalt buffer comprising a negatively charged detergent.
 15. A method forthe isolation of proteins from a solution containing one or moreproteins, comprising the following operations: a) contacting a solutioncontaining one or more proteins having a pH in the range of 2.0 to 10.0and a total salt content corresponding to an ionic strength of at themost 2.0 with a solid phase matrix comprising a functionalized matrixbackbone carrying a plurality of functional groups of the followingformulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinthe solution containing the proteins comprises a negatively chargeddetergent.
 16. A method for the isolation of proteins from a solutioncontaining one or more proteins, comprising the following operations: a)contacting a solution containing one or more proteins having a pH in therange of 2.0 to 10.0 and a total salt content corresponding to an ionicstrength of at the most 2.0 with a solid phase matrix comprising afunctionalized matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinthe molecular weight of the ligand-spacer arm (SP1-L) is below 250Dalton.
 17. A method for the isolation of proteins from a solutioncontaining one or more proteins, comprising the following operations: a)contacting a solution containing one or more proteins having a pH in therange of 2.0 to 10.0 and a total salt content corresponding to an ionicstrength of at the most 2.0 with a solid phase matrix comprising afunctionalized matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinL is an aromatic radical selected from the group consisting of a phenyl,1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2,3-benzenetriyl,1,2,4-benzcnetriyl, 1,3,5-benzenetriyl,1,2,3,4-benzenetetrayl, 1,2,3,5-benzenetetrayl, 1,2,4,6-benzenetetrayl,and 1,2,3,4,5-benzenepentayl.
 18. A method for the isolation of proteinsfrom a solution containing one or more proteins, comprising thefollowing operations: a) contacting a solution containing one or moreproteins having a pH in the range of 2.0 to 10.0 and a total saltcontent corresponding to an ionic strength of at the most 2.0 with asolid phase matrix comprising a functionalized matrix backbone carryinga plurality of functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinL is an aminobenzoic acid selected from the group consisting of2-amino-benzoic acid; 2-mercapto- benzoic acid; 3-aminobenzoic acid;4-aminobenzoic acid; 4-amino-2-chlorobenzoic acid; 2-amino-5-chlorobenzoic acid; 2-amino-4-chlorobenzoic acid;4-aminosalicylic acids; 5- aminosalicylic acids; 3,4-diaminobenzoicacids; 3,5-diaminobenzoic acid; 5-aminoisophthalic acid; and4-aminophthalic acid.
 19. A method for the isolation of proteins from asolution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 10.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinL is a heteroaromatic compound comprising a substituent selected fromthe group consisting of 2- amino-nicotinic acid, 2-mercapto-nicotinicacid, 6-amino-nicotinic acid and 2-amino- hydroxypyrimidine-carboxylicacid.
 20. A method for the isolation of proteins from a solutioncontaining one or more proteins, comprising the following operations: a)contacting a solution containing one or more proteins having a pH in therange of 2.0 to 10.0 and a total salt content corresponding to an ionicstrength of at the most 2.0 with a solid phase matrix comprising afunctionalized matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group having a pKa-value in the rangeof 1.0-6.0, with the proviso that the molecular weight of theligand-spacer arm (SP1-L) is at the most 500 Dalton; and the ligandconcentration is in the range of 10-120 μmol/ml hydrated, sedimentedsolid phase matrix; whereby at least a part of the proteins becomesbound to the solid phase matrix; then b) separating the solid phasematrix having proteins bound thereto from the solution; c) optionallywashing the solid phase matrix; and d) contacting the solid phase matrixwith an eluent in order to liberate one or more of the proteins from thesolid phase matrix, with the further proviso that the following criteriaare fulfilled: (i) the solid phase matrix has a binding efficiency of atleast 50% when tested at a pH in the range of 2.0 to 10.0 in theStandard Immunoglobulin Binding Test; and (ii) the stability of thesolid phase matrix in 1 M NaOH is such that incubation of the matrix in1 M NaOH in the dark at room temperature for 7 days reduces the bindingefficiency at a pH in the range of one pH unit lower than the bindingmaximum pH value to one pH unit higher than the binding maximum pHvalue, as determined in the Standard Immunoglobulin Binding Test, byless than 25 % compared to a corresponding non-incubated matrix, whereinthe spacer SP1 is a short chain aliphatic biradical selected from thegroup of consisting of -CH₂-CH(OH)-CH₂-, -(CH₂)₃-O-CH₂-[-]CH(OH)-CH₂-,or -CH₂-CH(OH)-CH₂-O-(CH₂)₄-O-CH₂-CH(OH)-CH₂-.
 21. A method according toclaims 12, 13, 14, 15 or 20, wherein L is an aromatic radical selectedfrom benzene radicals and naphthalene radicals.
 22. A method accordingto claims 12, 13, 14, 15, 16, 17, 18, 19 or 20, wherein the acidic groupis selected from a carboxylic acid group (-COOH), a sulfonic acid group(-SO₂OH), sulfinic acid group (-S(O)OH), phosphinic acid group(-PH(O)(OH)), phosphonic acid monoester groups (-P(O)(OH)(OR)), andphosphonic acid group (-P(O)(OH)₂).
 23. A method according to claims 12,13, 14, 15, 16, 17, 18 or 20, wherein the molecular weight of theligand-spacer arm (SP1-L) is below 250 Dalton.
 24. A method for theisolation of proteins from a solution containing one or more proteins,comprising the following operations: a) contacting a solution containingone or more proteins having a pH in the range of 2.0 to 6.0 and a totalsalt content corresponding to an ionic strength of at the most 2.0 witha solid phase matrix comprising a functionalized matrix backbonecarrying a plurality of functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein the pH of the eluent is in the range of 6.0 to
 11. 25. Amethod for the isolation of proteins from a solution containing one ormore proteins, comprising the following operations: a) contacting asolution containing one or more proteins having a pH in the range of 2.0to 6.0 and a total salt content corresponding to an ionic strength of atthe most 2.0 with a solid phase matrix comprising a functionalizedmatrix backbone carrying a plurality of functional groups of thefollowing formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein the washing of the solid phase matrix (operation (c))comprises washing with an aqueous solution comprising a negativelycharged detergent.
 26. A method according to claim 25, wherein thewashing of the solid phase matrix (operation (c)) comprises washing withan inorganic or organic salt buffer comprising a negatively chargeddetergent.
 27. A method for the isolation of proteins from a solutioncontaining one or more proteins, comprising the following operations: a)contacting a solution containing one or more proteins having a pH in therange of 2.0 to 6.0 and a total salt content corresponding to an ionicstrength of at the most 2.0 with a solid phase matrix comprising afunctionalized matrix backbone carrying a plurality of functional groupsof the following formulaM-SP1 -L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety; wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein the solution containing the proteins comprises anegatively charged detergent.
 28. A method for the isolation of proteinsfrom a solution containing one or more proteins, comprising thefollowing operations: a) contacting a solution containing one or moreproteins having a pH in the range of 2.0 to 6.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1 -L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety, wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein the molecular weight of the ligand-spacer arm (SP1-L) isbelow 250 Dalton.
 29. A method for the isolation of proteins from asolution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 6.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety, wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein L is an aromatic radical selected from the groupconsisting of a phenyl, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene,1,2,3-benzcnetriyl, 1,2,4-benzcnetriyl, 1,3,5-benzenetriyl,1,2,3,4-benzenetetrayl, 1,2,3,5-benzenetetrayl, 1,2,4,6-benzenetetrayl,and 1,2,3,4,5-benzenepentayl.
 30. A method for the isolation of proteinsfrom a solution containing one or more proteins, comprising thefollowing operations: a) contacting a solution containing one or moreproteins having a pH in the range of 2.0 to 6.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety, wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix; whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein L is an aminobenzoic acid selected from the groupconsisting of 2-amino-benzoic acid; 2-mercapto- benzoic acid;3-aminobenzoic acid; 4-aminobenzoic acid; 4-amino-2-chlorobenzoic acid;2- amino-5-chlorobenzoic acid; 2-amino-4-chlorobenzoic acid;4-aminosalicylic acids; 5- aminosalicylic acids; 3,4-diaminobenzoicacids; 3,5-diaminobenzoic acid; 5-aminoisophthalic acid; and4-aminophthalic acid.
 31. A method for the isolation of proteins from asolution containing one or more proteins, comprising the followingoperations: a) contacting a solution containing one or more proteinshaving a pH in the range of 2.0 to 6.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety, wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group, with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix, whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein L is a heteroaromatic compound comprising a substituentselected from the group consisting of 2- amino-nicotinic acid,2-mercapto-nicotinic acid, 6-amino-nicotinic acid and 2-amino-hydroxypyrimidine-carboxylic acid.
 32. A method for the isolation ofproteins from a solution containing one or more proteins, comprising thefollowing operations: a) contacting a solution containing one or moreproteins having a pH in the range of 2.0 to 6.0 and a total salt contentcorresponding to an ionic strength of at the most 2.0 with a solid phasematrix comprising a functionalized matrix backbone carrying a pluralityof functional groups of the following formulaM-SP1-L wherein M designates the matrix backbone; SP1 designates aspacer; L designates a mono- or bicyclic optionally substituted aromaticor heteroaromatic moiety, wherein the aromatic or heteroaromatic moietyand/or SP1 is carrying an acidic group; with the proviso that themolecular weight of the ligand-spacer arm (SP1-L) is at the most 500Dalton; and the ligand concentration is in the range of 10-120 μmol/mlhydrated, sedimented solid phase matrix, whereby at least a part of theproteins becomes bound to the solid phase matrix; then b) separating thesolid phase matrix having proteins bound thereto from the solution; c)optionally washing the solid phase matrix; and d) contacting the solidphase matrix with an eluent in order to liberate one or more of theproteins from the solid phase matrix, with the further proviso that thefollowing criteria are fulfilled: (i) the solid phase matrix has abinding efficiency of at least 50% when tested at a pH in the range of2.0 to 6.0 in the Standard Immunoglobulin Binding Test; and (ii) thestability of the solid phase matrix in 1 M NaOH is such that incubationof the matrix in 1 M NaOH in the dark at room temperature for 7 daysreduces the binding efficiency at a pH in the range of one pH unit lowerthan the binding maximum pH value to one pH unit higher than the bindingmaximum pH value, as determined in the Standard Immunoglobulin BindingTest, by less than 25 % compared to a corresponding non-incubatedmatrix, wherein the spacer SP1 is a short chain aliphatic biradicalselected from the group of consisting of -CH₂-CH(OH)-CH₂-, -(CH₂)₃-O-CH₂-[-]CH(OH)-CH₂-, or -CH₂-CH(OH)-CH₂-O-(CH₂)₄-O-CH₂-CH(OH)-CH_(2.) 33.A method according to claims 24, 25, 26, 27, 28 or 32, wherein L is anaromatic radical selected from benzene radicals and naphthaleneradicals.
 34. A method according to claims 24, 25, 26, 27, 28, 29, 30,31 or 32, wherein the acidic group is selected from a carboxylic acidgroup (-COOH), a sulfonic acid group (-SO₂OH), sulfinic acid group(-S(O)OH), phosphinic acid group (-PH(O)(OH)), phosphonic acid monoestergroups (-P(O)(OH)(OR)), and phosphonic acid group (-P(O)(OH)₂).
 35. Amethod according to claims 24, 25, 26, 27, 29, 30, 31 or 32, wherein themolecular weight of the ligand-spacer arm (SP1-L) is below 250 Dalton.36. A method according to claims 24, 25, 26, 27, 28, 29, 30, 31 or 32,wherein the acidic group has a pKa-value in the range of 1.0-6.0.